Acceptor substrates flexibility of previously characterized flavonol 7-O-rhamnosyltransferase (AtUGT89C1) from Arabidopsis thaliana was explored with an endogenous nucleotide diphosphate sugar and five different classes of flavonoids (flavonols, flavones, flavanones, chalcone and stilbenes) through a biotransformation approach. In contrast to the previous reports, this study highlights the expanded acceptor substrate promiscuity of AtUGT89C1 for the regiospecific glycosylation of diverse class of flavonoids at 7-hydroxyl position using microbial thymidine diphosphate (TDP)-L-rhamnose as sugar donor instead of uridine diphosphate-L-rhamnose. We examine the biocatalytic potential of AtUGT89C1 using endogenous sugar (TDP-L-rhamnose) from E. coli to generate a library of flavonoid 7-O-rhamnosides.