Objective To compare the cleaning effect of different ultrasonic cleaning time on ophthalmic microscopic instruments, and to explore the ultrasonic cleaning time that takes into account the cleaning quality and low loss. Methods A total of 30 used cataract kits and 30 used vitrectomy bags were selected, which were then averagely randomized into group A, B and C, with 180 ophthalmic microscopic instruments in each group. The three groups were all cleaned by using ultrasonic cleaning machine + automatic cleaning machine, and the ultrasonic cleaning time was 20 s, 40 s and 60 s, respectively. The cleaning quality of the three groups of cleaned instruments were evaluated by visual inspection, ATP fluorescence detection and protein residue detection. Results There weren′t statistically significant differences in qualification rates of the three groups according tovisual inspection and ATP fluorescence detection （both P＞0.05）,while it is significantly different according to protein residue detection among the three groups (P＜0.05), and there were statistically significant difference between Group A and Groups B and C (both P＜0.0167). Conclusion Ultrasonic cleaning + automatic cleaning machine is an effective way to clean ophthalmic microscopic instruments, and the appropriate ultrasonic time is 40-60 seconds.