Nitrate pollution is an increasingly serious problem in Chaohu Lake, China, with the rapid development of urbanization and industrialization surrounding the lake. The dual isotopic approach was employed to determine the sources of nitrate. Thirty-five and thirty-one water samples were collected in summer and winter, respectively. The concentrations of nitrate in the surface waters and lake waters were lower in summer than that in winter, due to the dilution of the wet deposition in summer. The concentrations of nitrate in the western part of the Chaohu Lake are higher than that in the eastern part, due to the inflow river discharge. The delta N-15 values of nitrate in winter are higher than that in summer, which is consistent with the seasonal variation of nitrate. The primary nitrate sources in northwestern part of the Chaohu Lake are the input of soil organic nitrogen and sewage/manure effluents, while the soil organic nitrogen is the main source in the southwestern and eastern part of the Chaohu Lake. Meanwhile, the data concerning delta O-18-NO3 (-) values in this study indicate that the nitrification process is one important source for the variability of nitrate in Chaohu Lake. This study can provide useful scientific guidance for the remediation and management of Chaohu Lake.