The Chapel of the Holy Bush is the most ancient shrine in the monastery, and it was around this site that the first community of Sinai anchorites gathered. The bush was mentioned by Egeria, who came to Sinai in 383-384 AC. The chapel is standing at the eastern end of the great basilica. Pilgrims enter this most holy place without shoes, in keeping with God’s command to Moses.

According to the Fathers of the Church, the bush that burned without being consumed by the flames was a type and prefiguration of the All-holy Theotokos, who bore within her the fire of the Godhead without being consumed and remained Virgin after Christ's birth. The feast of the Annunciation is celebrated here, and the first hymn of vespers on that feast makes a specific reference to the Burning Bush. Members of the community are tonsured here as well. The Divine Liturgy is celebrated in the Chapel of the Burning Bush on Saturdays.

The chapel is distinctive in not having an iconostasion separating the holy table from the rest of the chapel. The holy table is supported by four columns, allowing pilgrims to kneel and venerate. The apse above the holy table is decorated with a mosaic of rich materials but simple design. The walls are decorated with Iznik blue and white tiles, and covered with a multitude of beautiful icons.