The identification of non-point nitrate sources is important for the study and management of non-point pollution sources in agricultural areas. In this study, the nitrogen and oxygen dual-isotope technique and the Stable Isotope Analysis in R (SIAR) model were used to qualitatively and quantitatively analyze nitrate sources in the Huaihe River basin in China. The results showed that the inorganic nitrogen in the water was mainly in the form of nitrate nitrogen with a mean concentration of 1.32 mg/L. In the middle section, the major pollution sources were manure and domestic sewage (43.46%) and soil (30.23%), followed by chemical fertilizer (14.56%), dead branches and fallen leaves (8.18%), and atmospheric deposition (3.55%). Chemical fertilizers due to farmland cultivation were a bigger pollution source in the upper reaches (28.73%) than in the middle reaches (5.76%) and downstream (4.37%). Chemical fertilizers (74.86%) were the main pollution source on the south bank, while soil (34.43%), manure and domestic sewage (33.24%), and chemical fertilizers (29.31%) were the main pollution sources on the north bank. The main sources of NO3- were soil, chemical fertilizer, excrement and domestic sewage. Atmospheric deposition and dead leaves were the secondary sources of nitrate pollution. Major pollution sources comprised a large proportion of the total pollution (89.67%) compared to secondary pollution sources (10.33%). These findings highlight the significant impacts of manure, domestic sewage, and agricultural fertilizer, and they can be used to support nitrogen management practices in agricultural areas.