Flavonoids have gained much attention for their proposed positive effects for human health. Glycosylation is a significant method for the structural modification of various flavanols, resulting in glycosides with increased solubility, stability, and bioavailability compared with the corresponding aglycone. Natural product glycosylation by using enzymes has emerged as a topic of interest as it offers a sustainable and economical alternative source so as to address supply scalability limitations associated with plant-based production. Quercetin-3,4 '-O-diglucoside, as one of the major but trace bioactive flavonoids in onion (Allium cepa), is superior or at least equal to quercetin aglycone in its bioavailability. In the present study, the onion-derived enzyme, UGT73G1, coupled with sucrose synthase, StSUS1, from Solanum tuberosum formed a circulatory system to produce quercetin-3,4 '-O-diglucoside from quercetin, which preferred sucrose as a sugar donor and quercetin as a sugar acceptor. The optimal conditions were determined in order to increase the production of quercetin-3,4 '-O-diglucoside. The maximum concentration of quercetin-3,4 '-O-diglucoside achieved in a 10-mL reaction was 427.11 mg/L, from the conversion of 1 g/L of quercetin for 16 h at 40 degrees C and pH 7.2.