Objective To compare the effect of two cycle modes of automated endoscope reprocessor (AER) on the reprocessing of flexible endoscopes, and provide evidence-based guidance for selecting the optimal cleaning and disinfection protocol in clinical practice. Methods A total of 206 endoscopes after gastrointestinal endoscopy examination in the gastrointestinal endoscopy center of a tertiary first-class hospital in Jiangxi Province from June to October 2024 were selected and randomly divided into two groups using a random number table: group A (standard cycle mode, n=103) and group B (no-cleaning cycle mode, n=103). All endoscopes underwent standardized processing (bedside preprocessing-transfer-side leakage detection-manual cleaning-rinsing-visual inspection), then were reprocessed using different cycle modes of AER accordingly. After AER treatment, microbial sampling and culture were conducted on the endoscope biopsy channel, water/gas channel, and auxiliary water delivery channel respectively. The primary observation indicators included disinfection qualified rate, reprocessing time, as well as cleaning and disinfection costs. Results The qualified rates of disinfection for groups A and B were 96.12% and 92.23%, respectively, with no statistically significant difference (χ²=1.42, P>0.05). The single-endoscope reprocessing times for groups A and B were (46.98±0.64) and (34.02±0.66) minutes, respectively, with statistically significant difference (t=143.65, P<0.001). The unit costs for groups A and B were 45.81 and 42.65 Yuan, respectively, with cost-effectiveness ratios (C/E) of 49.09 and 47.63. Incremental cost-effectiveness analysis revealed that group A required an additional investment of 83.67 Yuan per 1% increase in qualified rate (ICER [incremental cost-effectiveness ratio]=83.67). Microbial culture results showed that the detected bacterial species in both groups were essentially the same, primarily including opportunistic pathogens such as Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Pseudomonas putida, and Enterobacter aerogenes. Conclusion On the basis of standardized manual cleaning, there is no significant difference in the reprocessing effectiveness of flexible endoscopes between the two circle modes of AER. The additional cleaning provided in the standard circle mode offers limited safety levels. All medical institutions at various levels should attach great importance to manual cleaning before placing endoscopes into the AER. Compared with the standard cycle mode, the absence of cleaning cycle in the AER can save time in endoscope reprocessing and reduce cleaning and disinfection costs. Sticking to standardized manual cleaning procedures, medical institutions at all levels may choose the no-cleaning cycle mode after comprehensively considering cost factors and endoscope turnover requirements.