In this study, we simultaneously investigated the community structure and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in the Grand Canal (the Zhenjiang section). Both clone library and qPCR (quantitative polymerase chain reaction) indicated that the abundance and diversity of AOB were higher than AOA in the Grand Canal for all four seasons. Among the 109 archaeal amoA sequences retrieved, 62.39 and 37.61% fell within the Nitrosopumilus and Nitrososphaera clusters, respectively. The 128 bacterial amoA gene sequences obtained in this study were grouped with known AOB sequences in the Nitrosomonas and Nitrosospira genera, which occupied 81.25 and 18.75% of the AOB group, respectively. The AOA abundance was significantly and positively correlated with the NH4-N. The AOB abundance did not show significant correlations with the measured parameters. Obvious differences were observed for the AOA community compositions obtained from different seasons. The community structure of AOB changed slightly. It indicated that AOB seemed to play a more important role for the nitrification process than AOA in this environment, and was more adapted to this environment.