Nitrospira was enriched from activated sludge by using fed-batch cultivation, and its related kinetic characterization was studied. The results showed that Nitrospira could be successfully enriched from the activated sludge for a controlled nitrite concentration no higher than 2 mg·L-1. The maximum nitrate oxidation rate was 48.72 mg·(g·h)-1. The fluorescence for the in situ hybridization results showed that Nitrospira accounted for about 75% of the total biomass, while Nitrobacter accounted for only 0.1%. In addition, the kinetic parameters of Nitrospira at 20℃ were also investigated. The results showed that the optimum growth temperature for Nitrospira was 30-35℃. The temperature correction coefficient τN was 1.046. The nitrite half-saturation constant KS and oxygen half-saturation constant KO were (0.32±0.03)mg·L-1 and (1.52±0.09)mg·L-1, respectively. This study on the kinetic characterization of Nitrospira provided a theoretical foundation for the treatment plant design and process optimization.