Enzyme-linked immunosorbent assay (ELISA) by use of monoclonal antibodies (MAbs) is very useful and helpful for the detection and quantification of the specific bacteria like nitrifiers in a mixed bacterial habitat. In this study, seven monoclonal antibodies were raised from splenocytes of mice(BALB/c) that are specific for the surface antigen of the two kinds of nitrifying bacteria. Three were directed against Nitrosomonas europaea (IFO 14298) and four were directed against Nitrobacter winogradskyi (IFO 14297). Cross-reactivities of MAbs against other strains of nitrifying bacteria as well as some kinds of representative heterotrophic bacteria in activated sludge and biofilm were checked to determine the usefulness of MAbs. It was found that there were some strain specificities between the same genera of IFO and ATCC strain. By means of a competitive ELISA, correlation curves for quantifying nitrifying bacteria were developed in a pure culture. It was found that this monoclonal antibody method could be used as a quick and powerful tool for estimating and controlling the population of nitrifying bacteria.