The hyporheic zone is essential in retaining nutrients and regulating downstream nutrient fluxes, but little is known about microbial communities in this area. The community structure of ammonia-oxidizing and denitrifying bacteria were studied at the river-sediment interface along a transect across an urban river. Using denaturing gradient gel electrophoresis (DGGE) analysis, the study showed that the ammonia-oxidizing and denitrifying bacteria presented the highest diversity at the oxic/fluctuating position, due to the flexible variations of water level and oxygen content. At this position, phylogenetic analysis revealed that amoA sequences mainly grouped with Nitrosospira and Nitrosomonas, which were belonged to β-Proteobacteria. Meanwhile, for denitrifying bacteria, they were mainly belong to α-, β-, γ-Proteobacteria, distributed across four broad clusters. Most of nosZ genes were closely related to uncultured environmental clones, indicating a high level of species richness in the denitrifying bacterial population. Thus, the ammonia-oxidizing and denitrifying bacteria could coexist at the river-sediment interface, which might play an important role in nitrogen removal of eutrophic rivers.