Human activities have greatly increased the nitrogen (N) loading in rivers over the past century, and consequently, N pollution has become a severe problem in aquatic systems. Nitrate (NO₃^-) is the predominant form of dissolved N in aquatic environments. In the present study, we evaluated N pollution characteristics and identified N sources using an isotope tracing technique in the upper Han River, a tributary of the Yangtze River with a length of 925 km in China. The objectives of this study were to evaluate the variations of N concentrations and explore the N sources in different time periods and areas. The results revealed that NO₃^- had significantly higher concentrations in July (22.75±17.75 mg L^-1) than that in other sampling months (14.00±10.85 mg L^-1 in November, 13.70±11.55 mg L^-1 in January, 4.99±6.10 mg L^-1 in April), and it also presented significant spatial variations (p<0.05) along the riverine network. Isotope analysis indicated a rather large range of isotope values, implying that the NO₃^- in the upper Han River originated from different sources, primarily from sewage. There was a large overlap of δ¹⁵NO₃^- values between different sources during the growing season (April/July), demonstrating the various inputs of N sources. Our results revealed the degraded water quality and poor control of N run off into the river. With the assessment of temporal–spatial variation and sources of N, improved management practices can be implemented to protect water resource and avoid further water quality deterioration in the upper Han River.