The MPN method was used to enumerate ammonia-oxidizing bacteria (AOB) in water and sediments of several shallow lakes. The suitable incubation time, medium types and substrate (ammonium sulphate) concentrations were studied. The results showed that, MPN values increased with the incubation time, reaching a stable maximum at some time stages, which was 40 days in all the samples for MSF medium. Among the three media used (XZ-AOB、MSF、SW), MSF give the highest MPN value. In addition, ammonium sulphate concentration in medium was an important factor affecting MPN estimation of AOB. Compared to AOB in lake sediments, AOB in lake water was more sensitive to ammonium sulphate concentration.