The first Assumption church in Odessa appeared 20 years after the city's founding. A small Old Believer chapel was consecrated on July 7, 1814. In 1845, a plan for the construction of a three-altar Assumption church was approved, and 8 years later, in 1853, one of the main relics of southern Tsarist Russia, the miraculous "Kasperovskaya" icon of the Mother of God, was brought here for the first time. In 1854, this icon protected the city from the Anglo-French-Turkish fleet that entered the Odessa bay.

On July 17, 1855, Saint Innocent laid the foundation for a new three-altar Assumption church on Preobrazhenskaya Street. The main funds for the construction were provided by Odessa merchants the Cherepenikovs, and the architect was L. Oton.

The construction of the church lasted 14 years, and on April 12, 1869, Archbishop Dimitry of Kherson and Odessa consecrated the cathedral building.

On January 19, 1875, Archbishop Leonty of Kherson and Odessa consecrated the main altar of the lower church in honor of Saint Nicholas, the miracle worker and protector of sailors and travelers, and on January 30, the second altar, in honor of the Three Hierarchs: Basil the Great, Gregory the Theologian, and John Chrysostom. Later, residents of Moscow donated icons and liturgical items for the lower church of the Odessa Assumption Church.

In the lower church of the Assumption Cathedral, the relics of the righteous Jonah are kept, who, according to contemporaries, manifested many different forms of holiness. Father Jonah possessed the gift of prophecy and healing of spiritual and physical ailments. The cross with which he blessed the water is still kept in the cathedral and is one of the most revered relics.

The Assumption Cathedral of Odessa also houses an icon of Saint Innocent with a particle of his relics.

With the arrival of the Bolsheviks, the Assumption Cathedral, like other churches in the city, faced difficulties. The church was closed in the late 1930s.

In 1942, Romanian occupiers resumed the activities of the lower church, for which they had to pump out water for several days, as it was flooded by 1.5 meters. During the Great Patriotic War, the upper church suffered more than the lower one, as a German bomb hit the central dome. Nevertheless, the 56-meter bell tower of the church became a symbol of the struggle of the people of Odessa against the fascist occupiers. One story recounts that one night Soviet partisans raised a red flag on the bell tower, which the Romanians managed to remove only the next evening. A memorial plaque once reminded of this event on the wall of the church.

The restoration of the church after the war became possible thanks to the efforts of Archbishop Nikon, Metropolitan Boris, and Patriarch Alexy I Simansky.

In 2008, at the initiative and blessing of Metropolitan Agafangel of Odessa and Izmail, a major renovation of the building was carried out, the heating system was improved, and the church was adorned with new wall paintings. The designers of the paintings in the Upper church were Vladimir Borisov and Rostislav Antonov, and the paintings themselves were executed by artists Vitaly and Daniil Khomnitsky. Thanks to the efforts of Vladyka Agafangel, the Sunday school was improved. In 2010, with his blessing, the dome over the bell tower was gilded, and in 2012, the main dome.