Objective To explore the cleansing effect of Nitric Oxide (NO) sustained-release silica nanoparticles (short for NO sustained-release agent) on endoscopic biofilm and its clinical application. Methods A total of 160 clinical endoscopes were randomly divided into two groups: the cleansing agent group (80 pieces, disinfected with cleansing agents), NO group (80 pieces, disinfected with NO sustained-release agent). A biofilm model of Pseudomonas aeruginosa was constructed and used as the control for phosphate buffered solution (PBS) treatment. A biofilm model of Pseudomonas aeruginosa on the surface of endoscopic lumen was built first in vitro. Scanning electron microscopy was then used to observe the microstructure of biofilm after treatment with NO sustained-release agent. Viable counting method was used to evaluate the cleansing effect of NO sustained-release agent on biofilm. Finally, at the clinical level, the actual disinfection effect of NO sustained-release agent on clinical endoscopy was evaluated by detecting the protein residues, viable counting and adenosine triphosphate (ATP) biofluorescence detection. Results The scanning electron microscopy showed that the biofilm was intact in the model group, but scattered bacteria were observed on the biofilm surface in the NO group and the detergent group. Compared with the model group [(4.86±2.67)×106 (colony-forming units, CFU)/mL], the standard CFUs of the NO group [(1.37±0.61)×104 CFU/mL] and the detergent group [(1.31±0.21)×105 CFU/mL] were significantly lower (detergent group VS model group, P=0.009; NO group VS model group, P=0.008), and there was significant difference between the detergent group and the model group (t=9.53, P=0.000 6). The levels of residual proteins in the endoscopic lumens before and after the treatment were 8.03±1.47 mg/mL and 0.50±0.37 mg/mL in the NO group, 8.01±1.51 mg/mL and 0.91±0.52 mg/mL in the detergent group with significant difference (P<0.01), and the reduction effect of the NO group was more significant. The disinfection of NO group and cleaning agent group was within the qualifying range, but the ATP bioluminescence value, protein residue and colony count of NO group (78.56±42.59 RLU, 0.50±0.37 mg/mL, 7.55±4.56 CFU) were significantly lower than those of detergent agent group (120.80±54.00 RLU,0.91±0.52 mg/mL,11.50±4.75 CFU, P<0.01). Conclusion NO sustained-release agent can effectively clear endoscopic biofilm and further improve the disinfection effect on endoscopes, which may be of great significance for improving the effects on treatment and prognosis of patients.