Due to the flow variability and habitat heterogeneity, spatial variation of river food web structure has been well understood. However, disturbance from anthropogenic activities, especially urbanization or eutrophication, may influence the spatial dynamic in food web structure of rivers. Further understanding of this spatial variation will provide available information for scientific management of lotic ecosystems. In this study, we used stable isotope analysis to investigate the δ¹³C and δ¹⁵N signatures of primary food sources and fish (Siniperca chuatsi, Hemiculter bleekeri, Pelteobagrus fulvidraco, Carassius auratus) in Raohe River during the dry period. Based on δ¹⁵N values, trophic position of four predominant fish species were calculated in urbanized (Poyang Section) and less urbanized reaches(Haikou section), respectively. Differences in isotopic values and trophic position of fish were then compared to determine if these changes were related to urbanization and river habitats. Little variation in δ¹³C values of fish was observed between Poyang and Haikou areas, which could be reflected by the values of particulate organic matter and attached algae. However, markedly differences were examined in δ¹⁵N values of fish species from those sampling areas. δ¹⁵N values in S. chuatsi and H. bleekeri from Poyang section were significantly higher than those in Haikou section, but C. auratus and P. fulvidraco had much lower values in Poyang section. Moreover, S. chuatsi, H. bleekeri and C. auratus had higher trophic positions in Poyang section than those species in Haikou section except for P. fulvidraco. Our data suggested that δ¹⁵N values of basal energy sources and eutrophication caused by anthropogenic activities could be the important factors that affected the nitrogen isotopic signatures of fish in different areas of Raohe River.