The church, located on the third floor of the seminary, was painted by Moscow artist Chereidnikov with assistants and the famous Orenburg artist Lukian Popov. The walls depict the holy hierarchs in full height. Above the arch opposite the altar is the solemn Entry of the Lord Jesus Christ into Jerusalem. On August 26, 1884, the church, dedicated to the three hierarchs: Basil the Great, Gregory the Theologian, and John Chrysostom, was consecrated by Bishop Benjamin II, who conducted the first Liturgy there. The seminary church received numerous donations. One benefactor, wishing to remain anonymous, gifted a silver robe with a golden crown adorned with seven stones: three diamonds and three pink rubies for the icon of the Mother of God. He also donated a bell weighing 20 poods. The abbess of the Orenburg Assumption Monastery, Taissia, gifted a silver chalice, and the supervisor of the spiritual school, Nikolai Speransky, an analogion with an embroidered cover. Priest Ioann Razypninsky from the Orenburg Trinity Church, who served in the military Georgievsky Cathedral and the Protection Church of the village of Tashla, transferred several icons. The Three Hierarchs Church received many other donations from its benefactors.

The academic year 1917-1918 became the last for the seminary, as the building was occupied by Red Army troops. Later, it housed the Chkalov Flight School, relocated from Serpukhov, and since 1995, the municipal cadet corps named after I.I. Nepluev. Thanks to the efforts of Metropolitan Valentin of Orenburg and Buzuluk, part of the seminary building was returned to the diocese by the decision of the Orenburg City Council on June 15, 2007. After restoration, the Orenburg Spiritual Seminary was opened there. The lost frescoes of the church were restored based on original sketches preserved in the Russian State Historical Archive. Masters from the Palekh Icon Painting Workshop 'Lik' from the village of Palekh in the Ivanovo region were invited for the frescoes of the restored church. The head of the group of artists, Oleg Romanovich Shurkus, visited Mount Athos before starting the work. In March 2010, the icon painters began painting the ceiling of the refectory, in September they started on the central part, and by March 2011, they completed the work in the narthex.

In the altar of the church is an ancient icon 'The Agony in the Garden,' created by the famous Russian artist Vladimir Egorovich Makovsky, transferred from the Protection Cathedral. Previously, it was located in the Orenburg Kazan-Bogoroditsky Cathedral. There is also the Kazan Icon of the Mother of God in a metal robe and an icon of Saint Nicholas the Wonderworker, painted by the Oryol icon painter Archpriest Sergey Baranov, transferred by Bishop Iriney of Orenburg and Gai.

All activities of the seminary are centered around the church, which is its heart. Daily liturgical services and readings of prayer rules for students take place here. Anyone can come here for prayer. In 2014, the seminary celebrated its 130th anniversary since its founding.