Stream ecosystems are the main receivers of nutrient and organic carbon exported from terrestrial ecosystems. Stream ecosystems are profoundly influenced by the land used in the surrounding area. Anthropogenic activities increase the amount of nutrient and organic carbon in both stream and benthic biofilms, which are closely related to differences in the aerobic methane-oxidizing microorganisms (MOB) bacteria microbial communities. We studied MOB bacteria microbial communities in the North Canal, Beijing, China. The results showed that the main form of nitrogen was ammonium nitrogen (NH₄⁺-N) in the North Canal. There are not any significant differences of the NH₄⁺-N between summer and winter, whereas there are significant differences between midstream and downstream. Results of heatmap analysis were closely related to the difference of the NH₄⁺-N in spatial. Phylogenetic analysis indicated that most of the cloned pmoA sequences in the North Canal were similar to those sequences from activated sludge and wastewater. The effect of nitrogen on MOB bacteria in the North Canal mainly depends on the main form of nitrogen and the source of pollutants. Moreover, MOB bacteria in downstream of the North Canal, had a more closer and modular microbial network than midstream, where bacteria communities with tightly connected species had proven to be more vulnerable and sensitive to various disturbances.