Dam regulation directly affects the intensity of hydraulic exchange and redox conditions in the hyporheic zone by changing river flow and water level, thereby influencing the biogeochemical processes of nitrogen. However, the quantitative mechanism of spatial succession of microbial communities in this process remains unclear. This study takes the Mojiabao section of the Hun River in Shenyang, China, as the study area. During the dry season when the dam releases water, three monitoring wells (W1, W2, W3) were set up along the direction perpendicular to the riverbank to collect groundwater samples. While measuring changes in nitrogen trioxides, we explored the spatial distribution characteristics of nitrogen transformation and microbial communities through high-throughput sequencing and PCR amplification of 16S rRNA genes, combined with alpha and beta diversity analyses. The results showed that the dominant phyla were Proteobacteria, Bacteroidetes, and Firmicutes; at the class level, γ-Proteobacteria were the dominant class; at the genus level, the nearshore W1 well was enriched with Rhodoferax (aerobic nitrification), while the offshore W3 well was enriched with Aeromonas and Pseudomonas (denitrification). For every 50 m increase in vertical distance from the riverbank, the Shannon index increased by 0.5-0.8, with a significant increase in species richness and denitrification capacity. The aerobic-anaerobic gradient created by dam releases promotes the succession of microbial communities from nearshore γ-Proteobacteria (nitrification-dominant) to offshore α-Proteobacteria and Bacteroidetes (simultaneous nitrification and denitrification). This study elucidates the succession phenomenon of microbial communities in the subsurface flow zone under hydrodynamic control, and the microbial diversity indicators can be used in the analysis and assessment system of river ecological conditions.