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The Wuding River Basin is a first-class tributary of the Yellow River, and the quality of its water ecological environment has a profound impact on the ecological protection and high-quality development of the Yellow River Basin. In order to identify the source of nitrate pollution in the Wuding River Basin, surface water samples of the Wuding River were collected from 2019 to 2021, and the temporal and spatial distribution characteristics and influencing factors of nitrate concentration in surface water in the basin were explored. Nitrogen and oxygen isotope tracer technology and the MixSIAR model were used to qualitatively and quantitatively determine the sources of surface water nitrate and their contribution rates. The results showed that there were significant spatial and temporal differences in nitrate concentrations in the Wuding River Basin. In terms of time, the mean concentration of NO - 3 -N in surface water in the wet season was higher than that in the flat-water period; spatially, the mean concentration of NO - 3 -N in the downstream surface water was higher than that in the upstream. The spatial and temporal differences in surface water nitrate concentrations were mainly affected by rainfall runoff, soil types, and land use types. The main sources of nitrates in the surface water of the Wuding River Basin during the wet season were domestic sewage, manure, chemical fertilizers, and soil organic nitrogen, whose contribution rates were 43.3%, 27.6%, and 22.1%, respectively, and the contribution rate of precipitation was only 7.0%. There were differences in the contribution rate of nitrate pollution sources in surface water of different river sections. The contribution rate of soil nitrogen in the upstream was significantly higher than that in the downstream, which was 26.5%. The contribution rate of domestic sewage and manure in the downstream was significantly higher than that in the upstream, which was 48.9%. To provide a basis for the analysis of nitrate sources and pollution control in Wuding River and even rivers in arid and semi-arid regions.