Objective To discuss the safety and efficacy of simultaneous bilateral flexible ureteroscopy (SBFU) with holmium laser lithotripsy for bilateral renal or ureteral calculi. Methods The clinical data of 78 patients (51 males, 27 females; average age \[43±12\] years, range 22 to 74 years) who underwent SBFU with holmium laser lithotripsy for bilateral renal or ureteral calculi in our hospital between January 2004 and February 2013 were retrospectively reviewed. The mean number of stones per case was 3.0±0.5 (range 2 to 5), and the mean diameter of the largest stone was (2.5±1.2) cm (range 1.0 to 5.0 cm). The operation was conducted under intravenous anesthesia. The upper ureteral calculi was removed firstly by ureteroscopy with holmium laser when it existed. After the ureteral access sheath was placed, flexible ureteroscopy was inserted into the renal pelvis, and then a 200 μm holmium laser fiber was used for fragmentizing stones and the large pieces was removed by stone basket. The operation was considered successful if no residual stone existed or residual stones were less than 4 mm in diameter during postoperative examination. Clinical data including operation time, stone-free rate and complications were analyzed. Results The flexible ureteroscopy was successfully placed in all the 78 cases, and the average operation time per case was (74.0±40.4) min (range 40 to 312 min), with no bleeding, ureteral perforation, avulsion or rupture during operation. The success rate of stone fragmentation was 93.6% (146/156) and the stone-free rate was 86.0% (134/156). Seven cases (9.0%, 7/78) required a second stage flexible ureteroscopy with holmium laser lithotripsy. The postoperative fever (>38℃) was found in 2 cases (2.6%, 2/78). Conclusion SBFU with holmium laser lithotripsy is a safe and effective treatment for bilateral renal or ureteral calculi. This procedure is a reliable treatment option for bilateral renal or ureteral calculi.