In order to clean the endoscopic tube more effectively, a new double-headed endoscopic cleaning brush was used in this study. A total of 130 colonoscopies were selected from the Digestive Endoscopy Center of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine from June 2019 to August 2020. The colonoscopy cleaning sequence was marked with odd and even number. Colonoscopies marked with the odd number were assigned to the conventional group (n=65) which received back and forth cleaning with the single-headed endoscope cleaning brush. Colonoscopies marked with the even number were assigned to the experimental group (n=65) which received one-way cleaning with a nylon brush at the head and a dense non-woven brush at the tail. The cleaning methods for endoscopes were in accordance with Flexible Endoscopic Cleaning and Disinfection Technical Specification WS507—2016. ATP bioluminescence tests and bacterial quantitative cultures were applied to evaluate the cleaning effects of the two methods. The results showed that ATP relative light unit (RLU) decreased in both the experimental group and the conventional group [530.63 RLU (26-3 559 RLU) VS 270.87 RLU (20-1 415 RLU)] before and after cleaning the endoscope tube, showing significant difference between the two groups (Z=-2.894, P<0.05). After scrubbing, the positive rate of bacterial culture on the brush head was 86.2% (56/65) for the double-headed non-woven brush head, 46.2% (30/65) for the double-headed nylon brush head and 32.3% (21/65) for the single-headed nylon brush head with significant differences among the three groups (χ2=41.046,P<0.05). The cleaning effect of the new double-headed endoscopic cleaning brush is better than that of the conventional single-headed endoscopic cleaning brush for soft endoscope cleaning. Non-woven brush is better than nylon brush in eliminating bacteria.