Urban storm runoff, as the primary transport medium for nutrients entering urban rivers, contributes to urban water contamination. Accurate source identification is critical for controlling water pollution. Although some studies have used nitrate isotopic composition (δ15N-NO3- and δ18O-NO3-) to identify nitrate (NO3--N) in urban storm runoff, the relatively low frequency of collecting samples in surface runoff within a single functional area hinders the understanding of spatial variations and dynamic process of NO3--N sources over the runoff process. This study investigated the nitrogen (N) concentrations and analyzed dynamic changes of NO3--N sources in surface runoff in different urban functional areas, drainage pipeline runoff, and channels during the complete runoff process in Wuxi, east China. The results showed that N concentrations in pipeline runoff and channels were higher than those in surface runoff, indicating that high concentration of N pollutants were accumulated in drainage pipelines. Information of δ15N-NO3- and δ18O-NO3- suggested that the main NO3--N source varied between runoff stages. NO3--N contribution from atmospheric deposition decreased in the order: surface runoff (57%) > residential pipeline runoff (25%) > channels (14%), while the opposite trend was observed for the contributions from sewage, increasing from 10%, 26% to 39%. In urban storm runoff, more sewage, fertilizers, and soil N were carried into the surface runoff after 30% of cumulative runoff ratio and carried into pipeline runoff in the initial 25% of cumulative runoff ratio in the residential area. As the first attempt to identify nitrate sources over the cumulative runoff in different urban functional areas, this work expands our understanding of the primary nitrate source in urban storm runoff. The findings provide important insights for developing strategies to mitigate non-point source water pollution.