Protostane triterpenes, which are found in Alisma orientale, are tetracyclic triterpenes with distinctive pharmacological activities. The natural distribution of protostane triterpenes is limited mainly to members of the botanical family Alismataceae. Squalene epoxidase (SE) is the key rate-limiting enzyme in triterpene biosynthesis. In this study, we report the characterization of two SEs from A. orientale. AoSE1 and AoSE2 were expressed as fusion proteins in E. coli, and the purified proteins were used in functional research. In vitro enzyme assays showed that AoSE1 and AoSE2 catalyze the formation of oxidosqualene from squalene. Immunoassays revealed that the tubers contain the highest levels of AoSE1 and AoSE2. After MeJA induction, which is the main elicitor of triterpene biosynthesis, the contents of 2,3-oxidosqualene and alisol B 23-acetate increased by 1.96- and 2.53-fold, respectively. In addition, the expression of both AoSE proteins was significantly increased at four days after MeJA treatment. The contents of 2,3-oxidosqualene and alisol B 23-acetate were also positively correlated with AoSEs expression at different times after MeJA treatment. These results suggest that AoSE1 and AoSE2 are the key regulatory points in protostane triterpenes biosynthesis, and that MeJA regulates the biosynthesis of these compounds by increasing the expression of AoSE1 and AoSE2.