Human-perturbed estuaries are significant sources of atmospheric nitrous oxide (N₂O), yet the source partitioning among multiple N₂O production pathways, and its relationship to substrate composition is unknown, hindering our capacity to constrain N₂O sources for effective emission mitigation. In this study, we conducted a comprehensive set of ¹⁵N isotope tracer incubation experiments across the salinity gradient of the Pearl River Estuary (PRE), a large, heavily perturbed estuary and N₂O emission hotspot, to quantify multiple nitrogen transformation processes and their associated N₂O production. Our results reveal that diverse nitrogen transformation pathways jointly sustain elevated N₂O production in the PRE, with nitrite and nitrate reduction pathways as the dominant contributors. Furthermore, by examining the relationship between substrate concentrations and the contribution of each substrate to N₂O production, we developed a substrate-specific N₂O production index to evaluate the efficiency of N₂O generation per unit substrate. Interestingly, while urea oxidation was not a major N₂O source, its contribution to N₂O production exceeded its relative abundance in the nitrogen pool, pointing to a disproportionately important, yet previously overlooked, N₂O source in coastal waters. Integrating global datasets from both coastal and open ocean systems, we further find that N₂O production rates are primarily regulated by substrate availability and organic matter conditions, whereas N₂O yields are more strongly controlled by transformation pathways, with reductive pathways consistently exhibiting higher yields than oxidative ones. Collectively, these findings suggest that shifts in substrate composition can control coastal N₂O production potential by restructuring nitrogen transformation pathways and their associated yield patterns.