By the end of the 15th and the beginning of the 16th century, the wooden churches of the Boris and Gleb Monastery fell into decline. By the decree of Grand Prince Vasily III and at his expense, in 1522, the first stone church was laid on the site of the wooden church dedicated to the holy princes Boris and Gleb. Its construction was completed two years later, and on September 22, 1524, the church was consecrated. It is believed that it was built by the talented Rostov master Grigory Borisov.

The Cathedral of Boris and Gleb is a small four-pillar single-domed church, typical of Russian architecture of the 15th-16th centuries. Such cathedrals were often built in cities, and their simple appearance suited monastery structures well. The only decorations of the cathedral are the perspective portals, cornices, and archivolts. The smooth walls with low windows are divided by flat pilasters into three unequal parts. Above the four-pitched roof rises the dome on a powerful drum. To the east, three lowered altar apses adjoin the church.

The original appearance of the cathedral was altered by later reconstructions. In the summer of 1778, under Archimandrite Varlaam, it was decided to raise the drum and replace the dome with an "onion" shape. These works were completed in 1779, but the new dome disrupted the harmony of the structure. Subsequently, it was covered with wooden shingles, and in 1836, four wooden drums with domes were added.

In 1780, the roof of the church was replaced with a four-pitched one, hiding the decorative belt of kokoshniks and ancient windows. In 1925, architect P.D. Baranovsky discovered them and removed the later drums.

The windows of the cathedral were enlarged in the second half of the 17th century when large-scale works were carried out. At that time, large windows appeared above the portals.

The exterior appearance of the cathedral was also changed by the chapel of the Prophet Elijah, added in 1810. During its construction, the ancient porch and decorations of the western portal were destroyed.

The interior of the cathedral is simple: smooth walls, absence of pilasters, arches on four pillars. Light penetrates through narrow windows. The iconostasis, which once adjoined the eastern pillars, was gilded with red gold.

According to the monastery inventories of the 18th century, the church was not painted in the 16th century. In 1735, there was only an image of Boris and Gleb in the cathedral, and in 1779, the walls were covered with lime whitewash. In 1783, the church was painted, and in 1842, the iconostasis and walls were adorned with new paintings. In 1911, under the guidance of artist F.E. Egorov, the church was painted in the Byzantine style.

The walls and vaults of the church are covered with paintings on Gospel themes with ornamental frames. On the pillars and walls of the apses are depicted the Moscow metropolitans and other saints. The paintings are executed in an academic manner.

In 1956, architect B.A. Ognev discovered a 16th-century fresco in the arcosolium of the northern wall. It depicts the holy princes Boris and Gleb, the founders of the monastery Fyodor and Pavel, as well as Grand Prince Vladimir and the Rostov saint Leonty.