Triterpenoid saponins are crucial natural products widely distributed in various medicinal plants, with Dipsacus asperoides being particularly rich in these compounds. However, the glycosyltransferases responsible for the biosynthesis of α-hederin, one of the primary bioactive secondary metabolites in D. asperoides, have not been elucidated. In this study, transcriptomic and compound analyses revealed 359 differentially expressed genes associated with secondary metabolism, with 271 involved in triterpenoid saponin glycosylation. Through correlation analysis, 71 candidate glycosyltransferases were identified, and two novel glycosyltransferases were functionally characterized. It was shown that DaUGT121 catalyzes the conversion of hederagenin into cauloside A, while DaUGT103 acts as a cauloside A 1,2-rhamnosyltransferase transforming cauloside A into α-hederin. These findings illuminate the biosynthesis of triterpenoid saponins in D. asperoides, providing insights into the molecular mechanisms and offering novel tools for synthesizing natural products with diverse sugar moieties.