Objective To evaluate the cleaning quality of different cleaning methods for laparoscopic instruments, ensure the cleaning quality of instruments. Methods The used laparoscopic instruments were classified into two categories:non-lumen instruments and lumen instruments. Three cleaning methods, traditional manual cleaning (A), manual cleaning + ultrasonic cleaning (B), manual cleaning + automatic cleaning disinfector (C), were adopted respectively for treating instruments. Cleaning quality of instruments were detected with ATP bioluminescence assay. Results The qualified rates of laparoscopic non-lumen instruments cleaned by methods A, B, and C were 78.75%, 95.71%, and 96.00% respectively, difference was significant (χ²=16.453,P<0.001);qualified rate of methods B and C for cleaning non-lumen instruments was higher than that of method A (P<0.016), there was no significant difference between methods B and C (P>0.016). Qualified rates of laparoscopic lumen instruments cleaned by methods A, B, and C were 76.47%, 98.75%, and 91.55% respectively, difference was significant(χ²=21.087,P<0.001); qualified rates of methods B and C for cleaning lumen instruments were both higher than that of method A (P<0.016), there was no significant difference between methods B and C (P>0.016). Conclusion Effect of manual cleaning + ultrasonic cleaning and manual cleaning + automatic cleaning disinfector on used laparoscopic instruments are both better than that of traditional manual cleaning method, can effectively guarantee the cleaning quality of instruments and help to ensure the operation safety of patients.