<p>River-estuary continua are hotspots for nitrogen transformations; however, whether N₂O and N₂ transformations vary in parallel in response to environmental variations along these continua remains poorly understood. This study compared spatial variations and environmental controls of excess N₂O (ΔN₂O) and excess N₂ (ΔN₂) concentrations along an urban river-estuary continuum, combining multi-season measurements with metagenomic analysis of nitrogen-cycling functional genes. Both ΔN₂O (−0.61–188.78 nmol/L) and ΔN₂ (−127.38–155.66 µmol/L) peaked in the river-estuary transition zone, coinciding with high nutrient inputs and potentially prolonged water residence time. However, we found a clear decoupling between these two gases, evidenced by their insignificant correlation and spatially offset peaks. Machine learning analysis indicated their divergent environmental drivers; nitrate was more important for ΔN₂O, while temperature and dissolved organic carbon (DOC) were more critical for ΔN₂. DOC was positively correlated with the genetic potential ratio of N₂ production to fixation, and temperature was negatively linked to the genetic potential for N₂O production relative to reduction. Furthermore, upon entering the estuary, ΔN₂ concentrations declined more sharply than ΔN₂O, leading to widespread N₂ undersaturation (i.e., ΔN₂&lt;0). This undersaturation is consistent with reduced denitrification, reflected by decreased denitrifier abundance due to high salinity and sulfide levels, alongside persistent N₂ fixation. The slower decrease in ΔN₂O is consistent with reduced N₂O consumption and elevated ammonia-oxidizing archaea to ammonia-oxidizing bacteria ratio. Our findings highlight distinct biogeochemical controls on N₂O and N₂ dynamics, providing insights for improving nitrogen removal and mitigating N₂O emissions in aquatic ecosystems.</p>