First church
The first church was known as the Μεγάλη Ἐκκλησία (Megálē Ekklēsíā, "Great Church"), or in Latin "Magna Ecclesia". Inaugurated by Constantius II on 15 February 360, it was built next to the area where the imperial palace was being developed. The nearby Hagia Eirene ("Holy Peace") church was completed earlier and served as cathedral until the Hagia Sophia was completed. Both churches acted together as the principal churches of the Byzantine Empire.

The first church was chronicled by Socrates of Constantinople (380–440), who claimed that it was built by Constantine the Great. It was built as a traditional Latin colonnaded basilica with galleries and a wooden roof. It was preceded by an atrium. It was claimed to be one of the world's most outstanding monuments at the time.

The Patriarch of Constantinople John Chrysostom came into a conflict with Empress Aelia Eudoxia, wife of the emperor Arcadius, and was sent into exile on 20 June 404. During the subsequent riots, this first church was largely burned down. Nothing remains of the first church today.

Second church
A second church was ordered by Theodosius II, who inaugurated it on 10 October 415. The basilica with a wooden roof was built by architect Rufinus. A fire started during the tumult of the Nika Revolt burned the second Hagia Sophia to the ground on 13–14 January 532.

Several marble blocks from the second church survive to the present day. The blocks feature reliefs depicting 12 lambs — 12 apostles — among other remains. They were originally part of a monumental front entrance, and are now displayed in the garden of the current church. They were discovered in 1935 during an excavation of the western courtyard by A. M. Schneider. No further excavation works were undertaken for fear of harming the present Hagia Sophia building.

Third church (current structure)
On 23 February 532, only a few days after the destruction of the second basilica, Emperor Justinian I took the decision to build a third and entirely different basilica, larger and more majestic than its predecessors.

Justinian chose the physicist Isidore of Miletus and the mathematician Anthemius of Tralles as architects; Anthemius, however, died within the first year. The construction is described by the Byzantine historian Procopius' On Buildings (Peri ktismatōn, Latin: De aedificiis). The emperor had material brought from all over the empire, such as Hellenistic columns from the Temple of Artemis at Ephesus. Large stones were brought from far-away quarries: porphyry from Egypt, green marble from Thessaly, black stone from the Bosporus region, and yellow stone from Syria. More than ten thousand people were employed during this construction. This new church was immediately recognized as a major work of architecture, demonstrating the creative insights of the architects. They may have used the theories of Heron of Alexandria to be able to construct a huge dome over such a large open space. The emperor, together with the patriarch Eutychius, inaugurated the new basilica on 27 December 537 with much pomp. The mosaics inside the church were, however, only completed under the reign of Emperor Justin II (565–578).

Earthquakes in August 553 and on 14 December 557 caused cracks in the main dome and the eastern half-dome to appear. The main dome collapsed completely during an earthquake on 7 May 558, destroying the ambon, the altar, and the ciborium over it. The emperor ordered an immediate restoration. He entrusted it to Isidorus the Younger, nephew of Isidore of Miletus. This time he used lighter materials and elevated the dome by 6.25 metres (20.5 ft), thus giving the building its current interior height of 55.6 metres (182 ft). This reconstruction, giving the church its present sixth-century form, was completed in 562. The Byzantine poet Paul the Silentiary composed a long epic poem, still extant, known as Ekphrasis for the rededication of the basilica, presided over by Patriarch Eutychius, on 23 December 562.

Hagia Sophia was the seat of the Orthodox patriarch of Constantinople and a principal setting for Byzantine imperial ceremonies such as coronations. The basilica also offered asylum to wrongdoers.

In 726 the emperor Leo the Isaurian issued a series of edicts against the veneration of images, ordering the army to destroy all icons, ushering in the period of Byzantine iconoclasm. At that time, all religious pictures and statues were removed from Hagia Sophia. After a brief reprieve under Empress Irene (797–802), the iconoclasts made a comeback. Emperor Theophilus (829–842) was strongly influenced by Islamic art, which forbids graven images. He had a two-winged bronze door with his monograms installed at the southern entrance of the church.

The basilica suffered damage, first in a great fire in 859, and again in an earthquake on 8 January 869 that made a half-dome collapse. Emperor Basil I ordered the church to be repaired.

After the great earthquake of 25 October 989, which ruined the great dome of Hagia Sophia, the Byzantine emperor Basil II asked for the Armenian architect Trdat, creator of the great churches of Ani and Argina, to repair the dome. His main repairs were to the western arch and a portion of the dome. The extent of the church's destruction meant that reconstruction lasted six years. The church was re-opened on 13 May 994.

In his book De caerimoniis aulae Byzantinae ("Book of Ceremonies"), Emperor Constantine VII (913–919) wrote about all the details of the ceremonies held in Hagia Sophia by the emperor and the patriarch.

Upon the capture of Constantinople during the Fourth Crusade, the church was ransacked and desecrated by the Latin Christians. The Byzantine historian Niketas Choniates described the capture of Constantinople. Many reputed relics from the church, such as a stone from the tomb of Jesus, the Virgin Mary's milk, the shroud of Jesus, and bones of several saints, were sent to churches in the West and can be seen now in various museums in the West. During the Latin occupation of Constantinople (1204–1261) the church became a Roman Catholic cathedral. Baldwin I of Constantinople was crowned emperor on 16 May 1204 in Hagia Sophia, at a ceremony which closely followed Byzantine practices. Enrico Dandolo, the Doge of Venice who commanded the sack and invasion of the city by the Latin Crusaders in 1204, is buried inside the church. The tomb inscription carrying his name, which has become a part of the floor decoration, was spat upon by many of the angry Byzantines who recaptured Constantinople in 1261. However, restoration carried out during the period 1847–1849 cast doubt upon the authenticity of the doge's grave. It is more likely a symbolic burial site to keep alive his memory.

After the recapture in 1261 by the Byzantines, the church was in a dilapidated state. The four buttresses in the west were probably built during this time. In 1317, emperor Andronicus II ordered four new buttresses to be built in the eastern and northern parts of the church. After new cracks had developed in the dome after the earthquake of October 1344, several parts of the building collapsed on 19 May 1346. After that, the church remained closed until 1354, when repairs were undertaken by the architects Astras and Peralta.