Abstract: Huang-Qin is a traditional Chinese medicine with antiviral, antioxidant, and anti-inflammatory activities. Its major bioactive compounds are diverse flavone O-glucuronides and glucosides. Although three flavonoid O-glycosyltransferases have been identified from S. baicalensis, this information is not sufficient to elucidate the structural diversity of flavonoid glycosides. In this study, nine glycosyltransferase candidate genes were discovered from S. baicalensis by BLAST analysis and their functions were characterized after heterologous expression. Three new flavone O-glycosyltransferases were able to catalyze the formation of major compounds in S. baicalensis, including baicalin and wogonoside. These enzymes could also utilize exogenous flavones as sugar acceptors. This work further elucidates biosynthetic pathways for Scutellaria flavonoid O-glycosides.