Cyclocarya paliurus (Batalin) Iljinsk is a medicinal plant belonging to the Juglandaceae family, and its leaves are used for a traditional sweet herbal tea with bioactivity against obesity and hyperglycaemia in China. It contains various bioactive specialised metabolites, such as flavonoids, triterpenes and their glucosides, while no glycosyltransferases (GTs) have been reported in C. paliurus to date. Herein, we identified and cloned the first glucosyltransferase C. paliurus GT1. The expression profiles of C. paliurus GT1 showed very high expression in young leaves, callus and branches, but relatively low expression in old leaves and bark and no expression in root. The recombinant C. paliurus GT1 protein was heterologously expressed in Escherichia coli and exhibited catalytic activity towards multiple flavonoids favouring substrate- and regio-specific biosynthesis. Further enzyme assays indicated a preference for certain hydroxyl group glucosylation by C. paliurus GT1. C. paliurus GT1 actively catalysed the glucosylation of flavones and flavonols, but it was less active towards isoflavones, flavanones or triterpenes. C. paliurus GT1 was also able to catalyse the attachment of sugars to the thiol (S-) or amine (N-) sites on aromatic compounds but not on aliphatic compounds. Molecular docking and site-directed mutagenesis analyses indicated that A43F, V84P, and M201Y dramatically altered the regio-selectivity and activity, and the W283M mutation and deletion of the V309-D320 region enhanced the activity and the formation of disaccharides. Herein, we present the identification and characterization of the first multi-functional glucosyltransferase in C. paliurus and provide a basis for understanding the biosynthesis of flavonoid glucosides. C. paliurus GT1 could be utilized as a synthetic biology tool for the synthesis of O-, N-, or S-glucosylated natural/unnatural products.