Background and Aim Adenosine triphosphate (ATP) bioluminescence assay is widely adopted in the West to allow rapid evaluation of endoscopes for bacteriologic/biologic residue, but this practice is rarely adopted in Asia. In this continuous quality improvement program, we evaluated the utility of ATP in bacteriologic surveillance on endoscope reprocessing. Methods A total of 456 samples (304 ATP samples and 152 culture samples) of 38 flexible endoscopes were assessed after routine clinical use in a private hospital in Hong Kong. Endoscopes were assessed with an ATP system and bacterial cultures at different time points during the reprocessing. Results After pre‐cleaning, the ATP values ranged from 228 to 65 163 relative light units (RLU) through all endoscope types. After manual cleaning, ATP values were decreased to 7–81 RLU (median, 19 RLU) for endoscope surface and 3–671 RLU (median, 12 RLU) for channel rinsate. There was a significant reduction in ATP levels between pre‐cleaning and after manual cleaning. One of the 38 (2.6%) endoscopes (a duodenoscope) had an ATP value of 671 RLU from channel rinsate, which exceeded the benchmark for cleanliness of >200 RLU, and was sent back for re‐cleaning. All endoscopes cultured no bacteria after high‐level disinfection (HLD) by automated endoscope reprocessor (AER) and storage up to 24 h. ATP values were <200 RLU for all endoscopes after HLD and storage. Conclusions Adenosine triphosphate bioluminescence assay offers a rapid, practical, and cost‐effective alternative for detection of endoscope microbial residue as well as a routine monitoring tool for endoscope cleanliness in the clinical setting.