Nitrite (NO2−) oxidation is an essential step of biological nitrogen cycling in natural ecosystems, and is performed by chemolithoautotrophic nitrite-oxidizing bacteria (NOB). Although Nitrobacter and Nitrospira are regarded as representative NOB in nitrification systems, little attention has focused on kinetic characterisation of the coexistence of Nitrobacter and Nitrospira at various pH values. Here, we evaluate the substrate kinetics, biological mechanism and microbial community dynamics of an enrichment culture including Nitrobacter (17.5 ± 0.9%) and Nitrospira (7.2 ± 0.6%) in response to various pH constrains. Evaluation of the Monod equation at pH 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 showed that the enrichment had maximum rate (rmax) and maximum substrate affinity (KS) for NO2− oxidation at pH 7.0, which was also supported by the largest absolute abundance of Nitrobacter nxrA (5.26 × 107 copies per g wet sludge) and Nitrospira nxrB (1.975 × 109 copies per g wet sludge) genes. Moreover, the predominant species for the Nitrobacter-like nxrA were N. vulgaris and N. winogradskyi, while for the Nitrospira-like nxrB, the predominant species were N. japonica, N. calida and Ca. N. bockiana. Furthermore, the rmax was strongly and positively correlated with the abundance of the Nitrobacter nxrA or Nitrospira nxrB genes, or N. winogradsk, whereas KS was positively correlated with the abundance of Nitrobacter nxrA or Nitrospira nxrB genes or Ca. N. bockiana. Overall, this study could improve basis kinetic parameters and biological mechanism of NO2− oxidation in WWTPs. •Biological kinetics of coexisting Nitrobacter and Nitrospira to pH was evaluated.•PH exposure has significant impact on the activity and nitrite affinity of NOB.•The pH dependence of NOB was species abundance, rather than microbial composition.•The rmax was strongly positively related with nxrA, nxrB genes and N. winogradsk.•The KS was positively correlated with nxrA, nxrB genes, Ca.N.bockiana.