Groundwater nitrate pollution in freshwater lake basins threatens ecosystems and human health, yet its sources and drivers remain poorly quantified. This study investigates the nitrate sources variation, geochemical characteristics of nitrate-contaminated groundwater, and its associated human health risks in the sub-basins of five rivers draining the Poyang Lake, China, via hydrogeochemical analysis and integrated assessment approach. Results showed that 52% of groundwater samples exceeded the background value of nitrate. High-nitrate groundwater primarily occurred as Cl-Ca and Cl-Na water types, while low-nitrate waters were of HCO3-Ca and HCO3-Na. Nitrate sources and transformations were strongly affected by nitrification, were closely associated with artificial surfaces and farmland Manure/sewage (M&S, 46.9%) and soil nitrogen (SN, 31.4%) were quantified as the dominant nitrate sources, while nitrogen fertilizers (NF, 17%) and atmospheric deposition (AD, 4.8%) made relatively minor contributions. Reconstructedinitial isotopic signatures showed lower δ15N and δ18O than measured values, yet still were within the manure and sewage isotopic ranges, reinforcing identified sources. Variations of uncertainty in nitrate sources tracing process were revealed by UI90, with SN (average 0.67), M&S (average 0.62), CF (average 0.54), and AD (average 0.11), respectively. Infants and children were predicted to be more vulnerable to nitrate exposure, with hazard quotient (HQ) values exceeding 1 in 54% and 46% of cases, respectively. Spatially, groundwater from the sub-basins of Rao and Xin rivers exhibited the highest contamination levels, while the sub-basin of Fu River showed the lowest. These findings underscore a need for targeted mitigation strategies to protect freshwater lake basins globally.