Estuaries and bays are key zones of intense land-sea interaction, where nitrogen cycling processes are particularly complex. Biological nitrogen fixation (BNF) represents an important source of new nitrogen to the ocean; however, it is conventionally assumed that elevated dissolved inorganic nitrogen (DIN) concentrations suppress diazotrophic activity, leaving the occurrence patterns and regulatory mechanisms of BNF in eutrophic coastal waters poorly understood. This study focused on the Jiulong River Estuary–Xiamen Bay as the study area, integrating multi-cruise field observations with molecular biological analyses and ¹⁵N₂ tracer incubations to systematically investigate the diazotrophic community composition, N₂ fixation rates, and their environmental controls in this region. Results showed that non-cyanobacterial diazotrophs dominated the diazotrophic community in this nutrient-rich coastal system, with N₂ fixation exhibiting pronounced spatiotemporal heterogeneity that was jointly regulated by salinity, organic carbon availability, and particle-associated microenvironments. Notably, particle-associated N₂ fixation may make a substantial contribution to overall nitrogen fixation in this coastal system. These findings provide new insight into the occurrence of N₂ fixation and its ecological role in estuarine–bay systems.