The study presented the method for isolating the heterotrophic nitrifiers and the characterization of heterotrophic nitrification.Continuous tests via a membrane bioreactor (MBR) were operated under the controlled conditions to proliferate the nitrifiers. Heterotrophic nitrifying bacteria were isolated from the system in which the efficiency of total nitrogen(TN) removal was up to 80%. Since no autotrophic ammonium and nitrite oxidizers could be detected by fluorescence in situ hybridization(FISH), oxidized-N production was unlikely to be catalyzed by autotrophic nitrifiers during the heterotrophic nitrifiers’ isolation in this study. The batch test results indicate that the isolated heterotrophic bacteria were able to nitrify. After 3 weeks incubation, the efficiencies of the COD removal by the three isolated bacterial strains B1, B2, and B3 were 52.6%, 71.7%, and 77.7%, respectively. The efficiencies of the TN removal by B1, B2, and B3 were 35.6%, 61.2% and 68.7%, respectively.