The medicinal plant produces sweet-tasting cucurbitane-type mogrosides from the atypical triterpenoid precursor 2,3,22,23-dioxidosqualene (SDO), rather than the conventional 2,3-oxidosqualene (SQO). However, SDO formation in mogroside biosynthesis remains unclear. Here, we systematically characterized two squalene epoxidases (SgSQE1/2) through phylogenetic analysis, heterologous expression, subcellular localization, qRT-PCR, and alanine scanning studies. Both SQE1 and SQE2 exhibited squalene epoxidase activity, with SQE2 catalyzing SDO formation in yeast. We identified two critical catalytic residues governing epoxidation efficiency through mutagenesis. Both SQEs were localized in the ER, while expression profiling revealed a similar trend between expression and mogroside accumulation in fruits. In our study, we developed a genomically engineered strategy for heterologous SQE characterization. These results lay the foundation for the SQE catalytic reaction involved in mogroside biosynthesis, and provide gene resources and a feasible approach for triterpene metabolic engineering.