ObjectiveTo make up for the deficiencies of traditional microbial determination method in water, a standard method for detecting the adenosine triphosphate (ATP) content of microorganisms in water is established. This enriched the detection system of microbial indices in water and provides a rapid detection means, thereby enhancing the detection capability of water quality in laboratories.MethodsBased on the method of the American Society for Testing and Materials——Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water (ASTM D4012—23a), a standardized ATP determination procedure had been established. Multiple laboratories had verified the limit of detection, lower limit of determination, precision, and accuracy. This method had also been applied in the detection of the entire water supply process and ice slurry cleaning of water supply pipelines.ResultsThe verification result showed that the method detection limit was 0.170 pg/mL, the lower limit of determination was 0.680 pg/mL, the range of the relative standard deviation was from 0.6% to 17.5%, the range of relative error was from -5.7% to 8.6%, the range of adding standard recovery was from 105.7% to 125.4%, and the results of the blank test were all less than the method detection limit. The method application showed that the ATP standard method could reflect the changes in microorganisms during the water purification process, the characteristics of water age in the water supply pipeline network, and the degree of microbial contamination on the inner wall of the pipeline.ConclusionThe method has the characteristics of a wide detection range, large concentration span, accurate data, convenient steps, and low waste and environmental protection. It can reflect the differences in water quality, water purification processes, and water age. And it is suitable for the microbial total quantity monitoring of the entire water supply process. It has been officially released as a group standard ——Determination of Microorganisms Content in Water-Adenosine Triphosphate (ATP) Bioluminescence Method (T/SAWP 003—2024) and can serve as an effective supplement of the microbial indices determination system in water.