The ponds are important hydrological channels connecting the upstream hillslope and the downstream water bodies, and important hotspots for nitrogen processing. As the primary mechanism of efficient nitrogen removal, the potential of denitrification and nitrogen removal in ponds and its influential factors were rarely been documented. In this study, fourteen typical ponds of four types (tea garden pond, village pond, fish pond, and forested pond) in Lake Tianmu Basin in the hilly region of the upper reaches of Lake Taihu were selected as our study sites. The characteristics of water quality and sediment change in summer and autumn were monitored respectively, and the levels of dissolved denitrifying product-N₂ in the ponds were directly determined by membrane injection mass spectrometry (MIMS). The results showed that N₂ supersaturated concentration in the overlying water of 14 ponds ranged from 1.36 μmol/L to 28.35 μmol/L, with an average of (8.23±6.04) μmol/L. N₂ supersaturated concentration was (8.81±4.08) μmol/L in summer and (7.64±7.46) μmol/L in autumn, and the concentration of N₂ supersaturated in different ponds in summer is higher than that in autumn. The ΔN₂ was the highest in tea garden ponds, followed by village ponds and fish ponds, and the lowest in forested ponds. The mean value of denitrification rates of 14 ponds was (4.75±3.27) mmol/(m²·d), which was highest in tea garden ponds, followed by village ponds and fish ponds, and lowest in forested ponds. Correlation analysis showed that denitrification was significantly positively correlated with nitrate concentration, and significantly negatively correlated with dissolved oxygen. Denitrification was also significantly positively correlated with nitrate concentration in sediment, and negatively correlated with bulk density, indicating that denitrification in ponds was simultaneously regulated by substrate concentration, dissolved oxygen concentration, and sediment physical and chemical properties. Compared and validated with the traditional denitrification measurement method, the membrane injection mass spectrometry combined with the water-gas exchange flux model proves feasible to estimate the denitrification potential of water. It is convenient to operate, requires fewer samples, and has a fast determination speed, which is suitable for the determination of denitrification in a flooded environment.