The manufacturing processes of many electronic and medical products demand the use of high-quality water. Hence the water supply systems for these processes are required to be examined regularly for the presence of microorganisms and microbial biofilms. Among commonly used bacteria detection approaches, the ATP luminescence assay is a rapid, sensitive, and easy to perform method. The aim of this study is to investigate whether ATP regeneration from inorganic pyrophosphate, a product of the ATP luminescence assay, can stabilize the bioluminescence signals in ATP detection. ADPglc pyrophosphorylase (AGPPase), which catalyzes the synthesis of ATP from PPi in the presence of ADPglc, was selected because the system yields much lower luminescence background than the commercially available ATP sulfurylase/adenosine 5′-phosphosulfate (APS) system which was broadly used in pyrosequencing technology. The AGPPase-based assay could be used to measure both PPi and ATP quantitatively and shows 1.5- to 4.0-fold slight increases in a 10-min assay. The method could also be used to stabilize the luminescence signals in detection of Escherichia coli, Pseudomonas aeruginosa, and Bacillus cereus in either broth or biofilm. These findings suggest that the AGPPase-based ATP regeneration system will find many practical applications such as detection of bacterial biofilm in water pipelines.