To evaluate the safety and efficacy of the flexible vacuum-assisted ureteral access sheath combined with the single-use flexible ureteroscopy in the treatment of renal calculi. We prospectively analyzed the patients treated with single-use flexible ureteroscopy from January 2023 to December 2023, among which 113 cases were treated with flexible vacuum-assisted ureteral access sheath (FV-UAS group) and 113 cases were treated with conventional sheath (conventional sheath group). Patient data ofthe two groups were collected and recorded before and after operation. No significant differences were observed between the two groups regarding demographics and pre-operative clinical characteristics. The duration of the operation was significantly longer in the FV-UAS group (79.5 ± 6.8 min) compared to the conventional UAS group (65.2 ± 7.3 min). However, the stone free rate (SFR) at one day and one month post-operation in the FV-UAS group was 85.6% and 98.2%, respectively, significantly higher than that in the conventional UAS group (72.6%, 90.3%). Complications such as infection and urosepsis were more common in conventional UAS group. Notably, hospitalization costs were lower in the FV-UAS group. Flexible vacuum-assisted ureteral access sheath combined with flexible single-use ureteroscope is a feasible and effective method for treating renal calculi. It offers higher SFR and lower rate of infectious complications.