The stem and leaves of American ginseng are rich in protopanaxatriol-type, protopanaxadiol-type, and ocotillol-type ginsenosides, which have important pharmacological activities. Among them, ocotillol-type ginsenosides have emerged as promising candidates for anti-cancer therapy. However, the scarcity of ocotillol-type ginsenosides presents significant challenges in terms of cost and sustainability during the synthesis process. In this study, we utilized three key glycosidases (HaGH03, PF-bgl, and DT-bgl) as essential tools in conjunction with diverse biotransformation strategies to augment the enrichment of pseudoginsenoside F11, RT5, and ocotillol. The results demonstrate that the combination of specific macroporous resin and the whole-cell catalyst HaGH03 enables rapid separation of ginsenoside extracts from Panax quinquefolium stems and leaves (PQE), effectively enriching pseudoginsenoside F11. In addition, the engineered mutant PF-bglF327C/F223I significantly enhanced the catalytic activity and addressed substrate inhibition issues, facilitating the enrichment of pseudoginsenoside RT5. Furthermore, the synergistic utilization of PF-bglF327C/F223I and DT-bgl enabled the one-step effective enrichment of ocotillol. This study presents a novel approach for rapid extraction and conversion of ocotillol-type ginsenosides while providing a feasible combination strategy for obtaining other rare natural products in the future.