Ammonia oxidation is a key component of the marine nitrogen cycle, it is mediated by three major lineages: ammonia-oxidizing archaea (AOA), bacteria (AOB), and the complete ammonia-oxidizing bacteria (comammox). Emerging evidence shows the coexistence of all the three lineages in the estuarine environment, however, their respective contributions to ammonia oxidation and N₂O production remain unresolved. In this study, we combined ¹⁵NH₄⁺ isotope labeling, specific inhibitors, size-fractionation experiments, and marker gene analyses to investigate ammonia oxidation and N₂O production along the salinity gradient of the Jiulong Estuary (JLE), a heavily human-impacted system. Our results reveal distinct salinity-dependent patterns in both ammonia oxidation and N₂O production. AOB dominated ammonia oxidation in low-salinity and mixing zones (salinity < 20), despite their low relative abundance (< 5% of the total ammonia-oxidizing community). In contrast, AOA were numerically dominant throughout the estuary but contributed to ammonia oxidation and N₂O production only in the high-salinity zone. Comammox bacteria exhibited low activity overall, accounting for less than 20% of the total activity across estuarine conditions, yet still represented a considerable contributor to both ammonia oxidation and N₂O production. Collectively, our study establishes a quantitative framework for disentangling the contributions of AOA, AOB, and comammox bacteria to ammonia oxidation and N₂O production in estuarine systems, offering new insights into the niche partitioning of ammonia-oxidizing lineages and improving our understanding of N₂O sources in the coastal ocean.

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