This research developed an innovative approach to reveal nitrogen sources, transformation, and transport in large and complex river networks in the Taihu Lake basin using measurement of dual stable isotopes of nitrate. The spatial patterns of δ15N corresponded to the urbanization level, and the nitrogen cycle was associated with the hydrological regime at the basin level. During the high flow season of summer, nonpoint sources from fertilizer/soils and atmospheric deposition constituted the highest proportion of the total nitrogen load. The point sources from sewage/manure, with high ammonium concentrations and high δ15N and δ18O contents in the form of nitrate, accounted for the largest inputs among all sources during the low flow season of winter. Hot spot areas with heavy point source pollution were identified, and the pollutant transport routes were revealed. Nitrification occurred widely during the warm seasons, with decreased δ18O values; whereas great potential for denitrification existed during the low flow seasons of autumn and spring. The study showed that point source reduction could have effects over the short-term; however, long-term efforts to substantially control agriculture nonpoint sources are essential to eutrophication alleviation for the receiving lake, which clarifies the relationship between point and nonpoint source control.