A competitive immunosensor using a monoclonal antibody has been developed for the enumeration of Nitrobacter in activated sludge and other environmental samples. Its cross-reactivity was tested against a number of bacterial strains and isolates. All strains of the nitrite-oxidising genera Nitrobacter and Nitrococcus reacted strongly with the monoclonal antibody. The nitrite-oxidising Nitrospira moscoviensis, as well as the ammonia oxidising bacteria and the heterotrophic bacteria tested, did not show any affinity towards the antibody in the immunosensor. The numbers of Nitrobacter were analysed in sludge samples from several wastewater treatment plants in Sweden. Detectable amounts were found in all samples. This study shows the adequacy of using this immunosensor for the enumeration of Nitrobacter in natural environments.