Among more than 100 rice uridine diphosphate glycosyltransferases (UGTs), OsUGT-3 was selected as a candidate for producing flavonoid O-diglycosyltransferases based on phylogenetic analysis and molecular docking. This gene was functionally expressed in Escherichia coli. Analysis of kaempferol, luteolin, quercetin, and tricin reaction products using liquid chromatography-mass spectrometry revealed that these were diglucosylated. The glucosylation positions of kaempferol, which was the best substrate, were determined to be the 3- and 7-hydroxyl groups. This is the first flavonoid O-diglucosyltransferase described from rice.