A highly regio-specific and donor-promiscuous 3- O-glycosyltransferase, Sb3GT1 (UGT78B4), was discovered from Scutellaria baicalensis. Sb3GT1 could accept five sugar donors (UDP-Glc/-Gal/-GlcNAc/-Xyl/-Ara) to catalyze 3- O-glycosylation of 17 flavonols, and the conversion rates could be >98%. Five new glycosides were obtained by scaled-up enzymatic catalysis. Molecular modeling and site-directed mutagenesis revealed that G15 and P187 were critical catalytic residues for the donor promiscuity. Sb3GT1 could be a promising catalyst to increase structural diversity of flavonoid 3- O-glycosides.