Abstract: The aim was to develop the simple preparation method of mogroside IIIE, and to lay the foundation for the development of the mogroside sweeteners. In the present study, the glycosidase CPU-GH17, which can regio-selectively biosynthesize mogroside IIIE from mogroside V, was screened from the established library of glycosidases. Then, the soluble expression condition of CPU-GH17 in E. coli was exploited by investigating isopropyl β-D-thiogalactoside(IPTG)concentration, culture temperature and induction time, and 0. 4 mmol/L IPTG, 15 °C and 12 h was used as optimal condition. The result showed that mogroside V could be completely converted into mogroside IIIE under the conditions of pH 6. 0, 45 °C, 3 U/mL enzyme loading, 5 mg/mL substrate concentration for 20 h. In conclusion, a biosynthetic system for the regio-selective preparation of mogroside IIIE by recombinant CPU-GH17 was successfully established and verified at a preparative scale.