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Nitrate (NO 3 − ) pollution is of considerable concern because its threat to human health and ecosystems. Herein, we chose the Wen-Rui Tang River watershed, one of typical rural-urban watersheds in Southeast China, as a case study. According to the analyses based on hydro-chemical ions, land-use classification, dual nitrate isotopes ( δ 15 N-NO 3 − and δ 18 O-NO 3 − ), and a Markov Chain Monte Carlo mixing model, the knowledge of contributions of potential NO 3 − pollution sources was obtained. Results showed that the study river was highly polluted by nitrogen, especially the increased riverine NO 3 − . Variations in δ 15 N-NO 3 − and δ 18 O-NO 3 − revealed that microbial nitrification was the main transformation process of nitrogen in surface water of the watershed, while bacterial denitrification was unlikely to be an important process at the specific sampling period. The δ 15 N-NO 3 − vs. δ 18 O-NO 3 − diagram incorporating chloride content qualitatively identified the main contribution of riverine NO 3 − was from municipal sewage discharges. On the basis of land-use characteristics, we applied hierarchical cluster method for subgrouping 44 sampling sites into four classes, namely, classes I (ecological), II (industrial), III (agricultural), and IV (urban). The Markov Chain Monte Carlo mixing model was employed to calculate proportional contributions of NO 3 − sources and articulated predominantly municipal sewage (23.5 – 42.2%) sourced nitrate in all classes. In addition, river sediments (13.8 – 20.8%) and atmospheric deposition (12.7 – 19.6%) were identified as important pollution sources. This study demonstrated that the comprehensive isotope tracing approach can effectively quantify the nitrate pollution source contributions for surface waters across the rural-urban interface.