Nitrous oxide (N₂O) dynamics in river-dominated estuaries reflect the combined effects of freshwater mixing and internal nitrogen transformations, yet their relative importance among different seasons remains poorly resolved. We investigated the distribution of dissolved N₂O in the Changjiang Estuary in March (dry season) and May (flood season) 2025. In March, surface N₂O concentrations ranged from 8.77 to 13.38 nmol L⁻¹, with a mean of 10.70 ± 1.39 nmol L⁻¹. Concentrations were highest in Changjiang River Water and decreased seaward. The negative correlation between N₂O and salinity indicated that freshwater transport and estuarine mixing were the main controls on its distribution. Mean surface N₂O saturation and sea-to-air flux were 93.53 ± 4.78% and −1.03 ± 1.50 μmol m⁻² d⁻¹, respectively, indicating that the study area was a weak sink for atmospheric N₂O. In May, surface N₂O concentrations ranged from 8.78 to 10.40 nmol L⁻¹, with a mean of 9.45 ± 0.51 nmol L⁻¹. Surface concentrations varied within a narrower range than in March, while localized enrichment occurred in deeper waters. N₂O showed no significant relationship with salinity but was positively related to depth and nitrate and negatively related to dissolved oxygen, indicating weaker control by freshwater mixing and enhanced N₂O accumulation in deeper waters. Mean surface saturation increased to 105.29 ± 4.28%, and the sea-to-air flux was 1.28 ± 0.99 μmol m⁻² d⁻¹, indicating that the study area became a weak N₂O source. Metagenomic data showed a relatively high representation of nirK but a low representation of nosZ, together with elevated amo and hao markers at the station exhibiting bottom-water N₂O enrichment. These patterns supported concurrent genetic potential for nitrite reduction and ammonia oxidation, with comparatively limited potential for terminal N₂O reduction. Incubations further demonstrated concentration-dependent N₂O responses to hydroxylamine, enhanced by nitrite availability. These results support a seasonal shift from predominantly mixing-controlled N₂O distributions in March to stronger contributions from subsurface biogeochemical production in May, accompanied by a reversal from a weak atmospheric sink to a weak source.