Flavonol glycosides are secondary metabolites important for plant development and stress defense such as UV-B irradiation. UDP-glycosyltransferase (UGT) catalyzes the last step in the biosynthesis of flavonol glycosides. Eriobotrya japonica is abundant in flavonol glycosides, but UGTs responsible for accumulation of flavonol glycosides remain unknown. Here, 13 flavonol glycosides including monoglycosides and diglycosides were characterized in different tissues of loquat by LC-MS/MS. UV-B irradiation significantly increased the accumulation of four quercetin glycosides and two kaempferol glycosides in loquat fruit. Based on UGT gene family analysis, transcriptome analysis, enzyme assays of recombinant proteins as well as transient overexpression assays in Nicotiana benthamiana, three UGTs were identified, i.e. EjUGT78T4 as flavonol 3-O-galactosyltransferase, EjUGT78S3 as flavonol 3-O-glucosyltransferase, and EjUGT91AK7 as flavonol 1 → 6 rhamnosyltransferase. This work elucidates the formation of flavonol glycosides in loquat through UGT-mediated glycosylation.