Abstract: The Tuojia River basin, a typical agricultural catchment in the subtropics, was investigated in this study to clarify the spatial and temporal distribution characteristics of nitrogen and the related stable isotopes in water system of the catchment. The environmental factors influencing nitrogen distribution and the sources of nitrate nitrogen and sediment organic matter nitrogen were also determined. The concentrations of NH₄⁺-N and NO₃^--N in surface water were analyzed in a continuous monitoring experiment. At the same time, the characteristics of δ¹⁵N-NO₃^- in water and of δ¹⁵N-Org in sediments were determined. Results showed that the concentration of NO₃^--N was significantly higher than that of NH₄⁺-N in the river, with respective mean values of 1.62 mg·L^-1 and 0.90 mg·L^-1. Higher values occurred in June, August and winter periods. NH₄⁺-N concentrations in urban and farmland regions were significantly different (P < 0.05) from other areas and obviously higher than that in other water bodies. The concentrations of NO₃^--N in urban, farmland and forest areas were higher than in other regions, with lower values in reservoir areas. The order of seasonal variations in NH₄⁺-N concentration in mainstream and tributary flows was winter > spring > summer > autumn, while that of NO₃^--N concentration was winter > summer > autumn > spring in mainstream, and autumn > winter > summer > spring in tributary flows. The concentrations of NO₃^--N of mainstream and tributary flows were high but similar, and NH₄⁺-N concentration in tributary flow was higher than that in mainstream flow. At source and estuary, NO₃^--N concentration was higher than NH₄⁺-N concentration. Also, nitrogen concentration of at source was lower than that in estuaries. The distributions of δ¹⁵N values (δ¹⁵N-NO₃^-) in the river and δ¹⁵N in sediment organic matters (δ¹⁵N-Org) were respectively -19.87‰ to 8.11‰ and -0.69‰ to 6.51‰. While the highest value of δ¹⁵N-NO₃^- was in the reach Ⅲ, the lowest was in the reach Ⅳ. The difference in δ¹⁵N-NO₃^- among different reaches was small in November, but was obvious in January and February. While the highest value of δ¹⁵N-Org in river sediment was also in the reach, the lowest was in the reach Ⅰ. The variation trend in δ¹⁵N-Org in the reach Ⅲ and reach Ⅳ was consistent with change in research time. However, the lowest δ¹⁵N-Org was in January in the reach Ⅰ and Ⅱ. The research indicated that there was nitrogen pollution in Tuojia River basin, and exogenous nitrogen was the priority in the region. The main environmental factors that influenced water pollution in the region included domestic sewage, industrial wastewater, farmland nitrogen and livestock/poultry waste. In addition, the sources of nitrogen in water bodies and sediments were mainly soil organic matter, synthetic fertilizers and terrestrial organic matter. The source of nitrogen in river systems was consistent with the identified environmental factors that caused nitrogen pollution. The study of the distributions and sources of nitrogen in the basin provided scientific basis for controlling nitrogen loss in the catchment, guiding agricultural production and improving water environment in the study area.