Two efficient and selective glycosyltransferases were identified from Sophora japonica L. Sj3GT could regio-selectively catalyse 3-O-glucosylation of quercetin to produce isoquercitrin, and Sj6''RhaT could further catalyse its 6''-O-rhamnosylation to generate rutin. It is particularly noteworthy that Sj6''RhaT shows high sugar donor selectivity towards UDP-rhamnose.