Testosterone 17-O-glucoside (T-17-G) is a precursor of the anticancer compound 3'-acetylated testosterone 17-O-glucoside (3'-AT-G). The biosynthesis of T-17-G was achieved by the glycosyltransferase (GT)-mediated glycosylation of testosterone with UDP-Glc. The low availability of UDP-Glc was detrimental to the synthesis of T-17-G, thereby limiting the druggability studies of 3'-AT-G. Therefore, finding cheaper alternatives of sugar donors is particularly urgent for the biosynthesis of T-17-G. Here, we reported the biosynthesis of T-17-G based on the transglucosylation reaction and an alternative sugar donor 2-nitrophenyl beta-D-glucopyranoside (oNP-Glc) was thus determined. Specifically, a flavonoid glycosyltransferase OcUGT1 from Ornithogalum caudatum was used as a biocatalyst to test the reactivities of various sugar donors with testosterone. The results indicated that OcUGT1 was able to catalyze the glucosylation of testosterone with UDP-Glc, thereby forming T-17-G and a diglucoside. Also, the generation of T-17-G was achieved by OcUGT1-assisted transglucosylation between testosterone with oNP-Glc or 4-nitrophenyl beta-D-glucopyranoside (pNP-Glc). Moreover, the glucosylation efficiency of testosterone in the presence of oNP-Glc was found to be higher than that with UDP-Glc. This data, together with the high availability of oNP-Glc revealed its applicability as a promising alternative to UDP-Glc in the glucosylation of testosterone.