Groundwater enriched with reactive nitrogen is a significant yet poorly quantified source of nitrous oxide (N2O). Using N-15-tracing incubations, we quantified substrate specific N2O production rates mediated by ammonium (NH4+) and nitrate (NO3-) along a groundwater flow path with a steep redox gradient (dissolved oxygen [DO] concentrations decreased from 250 to 20 mu M) in the semi-arid Hetao Basin, China. Elevated N2O concentrations were observed at both oxic upstream and localized reducing downstream sites, with over 90% of samples exhibiting oversaturation and substantial variability (4.6-188 nM) in groundwater with low-DO (20-102 mu M). Contrary to expectations that NO3--driven heterotrophic denitrification dominates downstream reducing environments, NH4+-driven pathway emerged as the primary source, contributing 66% of gross production. This dominance persisted even under oxygen-depleted conditions, fueled by synergistic NH4+-mediated processes, including hydroxylamine oxidation and nitrifier denitrification, particularly in reducing zones enriched with bioavailable organic nitrogen. Concurrently, abundant organic electron donors facilitate complete heterotrophic denitrification, thereby suppressing the accumulation of NO3--derived N2O. Despite localized spatial heterogeneity, where NO3--driven heterotrophic denitrification prevailing (> 50%) only at specific high C/N ratio sites, the overall potential for NH4+-driven N2O production was an order of magnitude higher than that of NO3-. Our findings demonstrate that NH4+-mediated processes dictate N2O dynamics across the redox continuum, highlighting a critical historical underestimation of ammonia's role in subsurface N2O sources. Given this study focuses exclusively on the aqueous phase, future research must incorporate sediments-associated biofilm dynamics and specific NH4+-driven subpathways to comprehensively refine groundwater N2O budgets.