Asiaticoside, madecassoside, and asiaticoside B are pentacyclic triterpenoid saponins derived from Centella asiatica, known to possess remarkable in wound-healing and skin-protective properties. However, the key biosynthetic steps for these bioactive compounds remain unclear, hampering the role of heterologous biosynthesis in ensuring a stable supply of these clinically important products. Here, we identified and characterized two missing enzymes (UGT94M2 and UGT94BE1) in the asiaticoside, madecassoside and asiaticoside B biosynthetic pathway, which sequentially added glucosyl moiety of UDP-Glucose and UDP-Rhamnose to the monoglucosides of asiatic acid (1), madecassic acid (5) and terminolic acid (9) in C. asiatica at C-28, respectively. Additionally, the multienzyme one-pot reaction was used to elucidate the biosynthetic pathway and generate the asiaticoside (4), madecassoside (8), asiaticoside B (12) and intermediate compounds, and the conversion rates for asiatic acid, madecassic acid and terminolic acid with UGT73AD1, UGT94M2 and UGT94BE1 was 77 %, 84 % and 82 %, respectively. Notably, the reconstruction of asiaticoside, madecassoside and asiaticoside B was realized in N. benthamiana with the newly identified genes, as well as seven already known ones. This study presents an almost complete biosynthetic pathway for asiaticoside, madecassoside and asiaticoside B, enabling the heterologous biosynthesis of these pentacyclic triterpenoids in other organisms.