Nitrate (NO ) contamination of surface water is a global environmental problem that has serious consequences for watershed ecosystems and endangers human health. It is crucial to identify influences of different sources of NO , especially the incoming water from upper reaches. A combination of hydrochemistry and multi-isotope tracers (δ B, δ N-NO , and δ O-NO ) were used to determine NO sources and their transformation the North Jiulong River (NJLR), Southeast China. The findings revealed that NO , which accounted for an average of 87.1% of dissolved inorganic nitrogen (DIN), was the main chemical form of nitrogen species. The integration of dual stable isotopes of NO , δ B, and hydrochemistry showed that NO was primarily contributed by sewage, soil nitrogen (SN), and ammonium (NH ) via precipitation or fertilizers. The contributions from the sewage and soil nitrate source were almost equivalent and much higher than those from other sources in the NJLR watershed. The contributions from diverse sources varied seasonally and spatially. Manure and sewage (M&S) were the leading sources in the summer and autumn, accounting for 60.9 ± 8.5% and 47.3 ± 7.9%, respectively. However, NO fertilizers were the predominant source in the spring and winter. The NO inflow from upper reaches was proposed as an additional end-member to identify its contribution in the midstream and downstream in this study. The contributions of NO from the upper reaches were significant sources in the midstream and downstream, accounting for 27.2 ± 17.8% and 42.9 ± 21.9%, respectively. The obvious decline in local NO contribution shares from midstream to downstream implied structural changes in pollutant sources and regional environmental responsibility. Therefore, tracing nitrate sources and quantifying their contributions is critical for clarifying environmental responsibilities for precise local nitrogen management in watersheds.