In 1657, the first wooden church of the Assumption of the Blessed Virgin Mary was built in Kharkiv, located in the fortress just south of the modern Assumption Cathedral. It was small, cold, and poor, lacking liturgical books and having paper icons. As the city grew and new churches appeared, the Assumption Church could no longer accommodate all the parishioners. Between 1685 and 1687, the first stone cathedral was erected on its site, becoming the first in Kharkiv. Like most cathedrals, it had a cross-dome structure, and its roof and five domes were covered with oak shingles. Nearby stood a stone tent-like bell tower with two "chambers" and a cellar that were rented out, providing income for the church. On March 3, 1733, a fire destroyed the cathedral, leaving only the stone walls. A year later, it was restored, with the domes covered in white tin and the cross gilded, but soon cracks appeared in the building. In the 1870s, it was dismantled to build a new cathedral that would match Kharkiv's status as a trading and industrial center of the Sloboda-Ukraine province. The old church did not reflect the spirit of the Catherine era.

When designing the new church, the temple of St. Clement in Moscow, built between 1762 and 1770, was taken as a model. Its feature was a square outline in plan and the absence of semicircular apses. The central light drum of the dome rested on four pylons, and the corner domes did not connect with the interior space. The external appearance of the cathedral reflected the transition from Baroque to Classicism. The facades were restrained, with stucco decorations reminiscent of Baroque. The interiors were lavish, including a carved gilded iconostasis made according to Rastrelli's sketch.

The construction of the cathedral lasted twelve years and was completed in June 1783. The new cathedral was significantly larger than the old one and had four altars: the main Assumption altar, the southern altar in honor of the Kazan Mother of God, the northern altar in honor of St. Catherine, and another in the choir in honor of Peter and Paul. The pride of the cathedral were the icons of the Elets Mother of God and St. Nicholas. After the completion of the cathedral, the question of building a new bell tower arose, and a decision was made in 1819. A church in honor of Alexander Nevsky was arranged in the bell tower, in memory of the victory in the Patriotic War of 1812. Construction began on May 8, 1821, and continued with interruptions until 1833. In 1837, work resumed under the guidance of architect K.A. Ton. In 1841, a gilded cross weighing 400 kg and 5 m high was installed on the bell tower. The work was completed in 1844, and the bell tower became the tallest in the city, with 12 bells and a clock ordered from Paris. The bell tower, connected to the church, became the main dominant feature of the city center, combining Baroque and Classicism styles.

After the 1917 revolution, the cathedral and bell tower were used for purposes other than intended and suffered destruction. In 1973, restoration began, during which the foundations and walls were reinforced, the drums and dome were restored, and the finishing was updated. In 1986, the House of Organ and Chamber Music opened in the building.

On October 29, 2004, by order of the head of the Kharkiv regional administration E.P. Kushnarev, the complex of the Assumption Cathedral was transferred to the jurisdiction of the UOC, but the decision was challenged in court. In 2006, the cross on the bell tower was restored. In 2009, the cathedral was handed over to the Kharkiv Diocese of the UOC, and until 2010, concerts and services were held there. In 2016, after the opening of a new organ hall, the cathedral fully transitioned to the jurisdiction of the Diocese.