It is with the ascension of the Roman emperor Constantine that a new era began for Alexandria, as well as for the Empire as a whole. By defeating his co-ruler Licinius (Rome had begun the practice of having two rulers, one for the eastern half of the Empire, and one for the western half), Constantine became sole emperor. He created an eastern capital for the Empire in the city of Byzantium, which he renamed Constantinople (this would not be the last name change the city would go through, after the sack by the Ottoman Turks in 1453 it would be called Istanbul). His new capital, in which he spent much of his remaining life until his death in AD 337, was small but growing, but it was a far cry from the mighty city it would become under the Byzantines. Constantine imported Greek and Roman statuary to decorate the city, ordered the construction of buildings in the traditional Roman style, and had half the grain shipments from Alexandria shipped to Constantinople. Yet what Constantine is most noted for today was his policy towards the various religions in the Empire. He supported both the Roman religion as well as Christianity. Clerics of both faiths were exempted from taxation and having to serve on city councils (a move which prompted a great number of ordinations), the same financial help which had been given to the building of Roman temples was now shared with the Christians, and Constantine himself was responsible for the construction of the Church of the Holy Sepulchre at Jerusalem. Constantine also gave land and money to build a great church in Rome, which would later grow into the headquarters of the Christian religion: the Vatican.

With so much emphasis on Constantinople, and the fact that much of the Egyptian grain production was being shipped there, Alexandria began to slip from its position at the center of the Mediterranean world. Meanwhile the old Roman Empire crumbled under barbarian invasions and internal conflict, and the Byzantine Empire rose in its place. The center of the world moved to Constantinople, which under the Byzantines became a center for art, science, and religious and secular learning. Alexandria continued to influence the world, only more subtly now. In 529 the emperor Justinian closed the Academy of Athens, forbidding the teaching of what he called "pagan philosophy", yet Alexandria's schools remained open, teaching Atristotelian and Platonic philosophy well into the eighth century.

Alexandria also received another moment of glory during the Byzantine Era, as the Byzantines became rather infatuated with classical Greek culture that had been largely lost under the Romans, but well-preserved by the learned of Alexandria. Royal patronage of the arts and sciences had long disappeared, yet the poets, teachers, and scholars went on for their art's sake, supporting themselves through pedagogy and commissioned writing. But this was not to last. In the early seventh century the most successful Persian attack on the Byzantine Empire took both Jerusalem and Alexandria. The emperor Heraclius managed to beat back the Persians to the point of collapse but a new onslaught began, this time from the south. After battling the Persians, the Byzantine rulers had little hope of defeating the forces that came sweeping north from the deserts of Arabia. The final defeat of the Byzantine armies in 636 left Palestine and Syria open to conquest by the Arabs, and they spread like wildfire over northern Africa, eventually bringing Alexandria under their control in 642.