Polders are an important part of the landscape pattern of Dongting Lake, and the pollution of inorganic nitrogen in surface water has become one of the serious environmental problems in the polder area of Dongting Lake. Taking the Quyuan Polder section of Pingjiang River as the research area, stable isotope and hydrochemical analysis methods were used, and the MixSIAR model was combined to quantitatively identify the contribution of different pollution sources of nitrate in surface water in the study area. The results showed that inorganic nitrogen in surface water in the Quyuan Polder section of Pingjiang River mainly existed in the form of nitrate and ammonia nitrogen. Temporally, there was no significant difference in the concentrations of nitrite nitrogen between the rainy season and the non-rainy season, while the average concentration of ammonia nitrogen was higher in the rainy season than in the non-rainy season. Spatially, the concentration of ammonia nitrogen inside the polder was significantly higher than that outside, while the concentration of nitrate nitrogen was significantly lower than that outside. Nitrogen and oxygen isotope and MixSIAR model analysis results proved that fertilizers, soil organic nitrogen, aquaculture wastewater, manure, and sewage were the main sources of nitrate in surface water in the study area. The contribution of these nitrogen sources to nitrate in surface water in the rainy season were 33.02%, 32.58%, 19.42%, and 11.69%, respectively, and those in the non-rainy season were 26.73%, 31.24%, 21.46%, and 16.93%, respectively. Atmospheric deposition contributed only 3.5% to the sources of nitrate in surface water. The transformation process of nitrate in surface water in the study area was mainly nitrification. The study analyzed the sources of nitrate pollution in the Quyuan Polder section of Pingjiang River, providing a basis for water quality safety management and non-point source pollution control in the region.