Objective：To evaluate the clinical efficacy and safety of flexible ureteroscopic lithotripsy using 365 μm holmium laser for nephrolithiasis in this study. Methods：Patients who were diagnosed as nephrolithiasis and submitted in our center between July 2017 and July 2018，were randomized into two groups：60 cases for 365 μm group treated by flexible ureteroscopic lithotripsy using 365 μm holmium laser，and 60 cases for 200 μm group treated by conventional flexible ureteroscopic lithotripsy. The surgery-related indexes，the complications and biochemistry examinations between two groups were compared. Results：Compared with 200 μm group，the operation time for cases in 365 μm group were significantly shortened. No significance was observed in the length of hospitalization and the stone free rate between two groups. For safety，the complication rate was significantly low and biochemistry examinations were significantly improved in 365 μm group. In addition，the damage of flexible ureteroscopy and fiber were less in 365 μm group than in 200 μm group. Conclusion：Our study firstly reported the highly efficientcy of flexible ureteroscopic lithotripsy using 365 μm holmium laser for nephrolithiasis in this cohort，which significantly shortened the operation time for the stones larger than 2 cm. Moreover，the comparable safety and higher cost-effectiveness were observed in 365 μm group.