The effect of AOB/NOB in nitrifying sludge on nitrification characteristics was deeply investigated in this study. Firstly, activated sludge only with AOB and NOB was successfully cultivated in two same SBRs through 80 cycles, respectively. The main substrate in their inflow was single, during cultivation influencing factors were controlled and sludge was discharged at regular intervals. Secondly, the ratio of abundance between AOB sludge and NOB sludge was determined through experiment which was about 1:1. Finally, experiment results about nitrification characteristics showed that nitrosation rate, ammonia oxidation rate, nitrite oxidation rate and oxygen uptake rate were influenced by A/N in nitrifying sludge. A/N should be close to 1:0 for steady operation of partial nitrification; there was no obvious positive relationship between ammonia oxidation rate and proportion of AOB in nitrifying bacteria; A/N in conventional nitrification sludge for municipal sewage treatment should not less than 1:2; combined with online monitoring for oxygen uptake rate, when it became stable, the startup of partial nitrification could be considered as nearly achieved.