The ability to detect and enumerate the specific bacteria is essential for determining their fate in various biological processes. Although fluorescent in situ hybridization (FISH) technique using rRNA-targeted oligonucleotide probes may be considered as the most rapid method for identification of a single bacterial cell, it could not efficiently count cells due to the limitations of light microscopy. In this study, to make accurate estimates of the active nitrifying bacterial populations from activated sludge samples, applied FISH method using Luminescence Spectrometer LS50B was used. Enumeration of nitrifying bacteria by FISH using Luminescence Spectrometer can help in the optimization of the performance of biological nitrogen removal processes.