Objective To simulate the cleaning and disinfection process of endoscopes during the acceptance of new endoscopy center, find out the existing problems and put forward solutions. Methods Manual and automatic endoscope reprocessor(AER)washing methods were used to simulate the cleaning and disinfection process of endoscope during the acceptance process of new endoscopy center. Pure water used for end-rinse and endoscopes after cleaning and disinfection were performed microbial detection, according to detected results, causes for the unqualification were analyzed, the corresponding improvement was made. Results The qualified rates of pure water used for end-rinse of endoscopes before and after disinfection of pure water supply pipeline were 0% and 100% respectively, the median (quartile) of bacterial contamination were 200 (186, 213) and 1.5 (0, 6) CFU/100 mL respectively, with significant difference (P<0.01). The qualified rates of manual cleaned and disinfected endoscopes before and after adjusting enzyme concentration were 80.00% and 91.67% respectively. Before and after AER were disinfected, the qualified rates of water specimens from AER at on-load condition were 30% and 100% respectively, the median (quartile) of bacterial contamination were 97.5(8,175) and 4(1.75,6.00)CFU/100 mL respectively,with significant difference (P<0.01). After endoscope lumen with suspected biofilm formation was disinfected with 2 000 mg/L peracetic acid, the qualified rate was 100%. Conclusion Problems existed during the acceptance of new endoscopy center are non-disinfection of pure water supply pipeline, incorrect enzyme concentration for endoscopic cleaning, non-disinfection of AER, and formation of biofilm of endoscope lumen, after targeted improvement, these problems are solved. In order to ensure medical safety and quality, medical institutions should attach great importance to the acceptance before operation of new endoscopy center, and eliminate risk factors for unqualified endoscopy disinfection.