Objective To evaluate the effect of delayed cleaning on the cleaning and disinfection quality of gastroscopes after pre-treatment with different solutions. Methods According to the factorial design table, combination of the pre-treatment cleaning solutions (factor A) (including multi-enzyme cleaning solution [A₁], clean water [A₂]) and delayed cleaning durations (factor B) (including 0 minutes after pre-treatment [B₁], 30 minutes after pre-treatment [B₂], 1 hour after pre-treatment [B₃], and 3 hours after pre-treatment [B₄])yielded eight groups (A₁B₁, A₁B₂, A₁B₃, A₁B₄, A₂B₁, A₂B₂, A₂B₃, A₂B₄). According to the usage order of gastroscopes, 96 gastroscopes used in the digestive endoscopy center of a tertiary first-class hospital from May to September, 2023 were randomly assigned to each group by random number table method, with 12 gastroscopes in each group. Specimens were taken at four time points: after pre-treatment, before cleaning, after cleaning, and after disinfection. Due to instant clea-ning, no specimen before cleaning were taken from A₁B₁ and A₂B₁ groups, thus only 3 specimens were taken from these two groups each. Four specimens were taken from gastroscopes in the rest groups, resulting in 360 specimens in total. The internal condition of the biopsy cavity was observed through a cavity detector during each delayed cleaning period after pre-treatment, and specimens were taken at the subsequent reprocessing processes of the gastroscopes. The microbial conditions of the gastroscopes after pre-treatment, before cleaning, after cleaning, and after disinfection were compared. Results After pre-treatment with multi-enzyme cleaning solution and clean water, there was no statistically significant difference in microbiological detection result (P>0.05). The biopsy cavity remained moist during the delayed cleaning period. There was no statistically significant difference in the microbial detection results of factors A and B before and after delayed cleaning as well as after disinfection (all P>0.05). There was no interaction effect between factor A and B. The distribution of bacterial colonies and disinfection qualified rate of gastroscopes after pre-treatment with two cleaning solutions were also not statistically different (both P>0.05). Conclusion Delayed cleaning for 30 minutes, 1 hour, and 3 hours after pre-treatment does not affect the cleaning and disinfection quality of gastroscopes. When clinical demand is urgent, immediate cleaning should be carried out. However, a certain buffering time (no longer than 3 hours) before cleaning is acceptable, when cleaning and disinfection workload is heavy and timely cleaning cannot be carried out.