Denitrification is an important pathway for removing nitrogen loads from water bodies. Accurately and rapidly measuring the denitrification rate in water bodies is crucial for assessing the nitrogen removal potential of lakes, reservoirs, and rivers. To address the issues of existing methods, such as the acetylene inhibition method and the _¹⁵N isotope tracer method, which are unable to provide in situ rapid measurements of denitrification rates, and are costly and time-consuming, this study proposes a novel method for rapid in situ measurement of denitrification rates in water bodies based on nitrogen gas increment. The method involves in situ water sampling, purging, incubation, and measurement, enabling rapid field detection of total denitrification rates. This method was applied in May–June 2024 to conduct point-based continuous monitoring and bay-wide surveys of denitrification rates in the Pengxi River, a tributary of the Three Gorges Reservoir. A comparison with the acetylene inhibition method was performed, and the influence of various environmental factors on denitrification rates was analyzed. Results showed that the denitrification rates measured by this method were approximately seven times higher than the denitrification rates obtained using the acetylene inhibition method, with a significant correlation between the two. During the monitoring period, the denitrification rates in the Pengxi River were highest in the surface layer, followed by the bottom layer and then the middle layer. The surface layer denitrification rates were mainly influenced by algal concentrations, while those in the bottom layer were primarily affected by turbidity. This study has significant implications for the rapid in situ measurement of water body denitrification rates and for accurately understanding and evaluating the nitrogen removal potential of water bodies.