Objective To analyze the causes of exceeding standard of colony count of enteroscope after disinfection, and further improve the reprocessing level of flexible endoscope. Methods Data of the third quarter routine environmental hygiene monitoring of digestive endoscopy center of a hospital conducted in August 2021 were analyzed, causes of secondary contamination of enteroscope after disinfection were found out and rectified. Results The monitored four gastroscopes, two specimens of purified water, two specimens of in-use disinfectants, three specimens of environmental object surfaces and two specimens of hygienic hand disinfection of health care workers were all qualified; however, a large number of bacteria were detected from four enteroscopies, bacterial colony number were 252, 315, 273, and 268 CFU/piece respectively, all specimens were detected Rhizobium radiobacter, Micrococcus luteus, and Sphingomonas paucimobilis. Possible risk factors leading to the failure of enteroscope monitoring were identified, specimens of air from air spray gun, inside of air filter and air line of filter outlet were detected further, a large number of the above microorganisms were found out.After the replacement of air compressor, air filter and air line, the number of bacteria in air from air spray gun decreased obviously.The 4 unqualified enteroscopes were cleaned, disinfected and dried again, bacterial contamination was detected, all were all qualified. Conclusion Contamination of air line of the digestive endoscope cleaning workstation is the main cause for the excessive number of bacteria in the lumen of enteroscope after disinfection.It is suggested that air line maintenance and biological monitoring should be included in the corresponding guidelines, so as to reduce healthcare-associated infection.