The increasing global nitrogen input poses a significant threat to aquatic environments, particularly in agricultural watersheds, where intensive human activities and insufficient water protection infrastructure exacerbate the risk of nitrogen pollution. Accurate identification of nitrogen pollution sources and the associated transformation processes is essential for protecting watershed ecosystems. In this study, a combination of hydrochemical analysis, correlation and principal component analysis, and stable nitrate isotopes (δ15N-NO3− and δ18O-NO3−) were employed to trace nitrogen transport pathways and source contributions in both surface water and groundwater within a typical agricultural watershed. The results revealed the presence of nitrogen pollution, including total nitrogen (TN), ammonia nitrogen (NH3-N), and nitrate nitrogen (NO3−-N), with significant spatial and seasonal variations in both surface water and groundwater. The spatiotemporal evolution of hydrochemical indicators and nitrate isotope compositions highlighted multiple potential sources of nitrogen, including soil input, agricultural input, and manure and sewage input. The results from stable isotope analysis in an R (SIAR) model indicated that ammonium fertilizers (7.1~78.4%) and manure and sewage (2.6~69.7%) were the primary sources of nitrates in surface water, while manure and sewage were the main sources in groundwater (67.9~73.7%). This research demonstrated that nitrification, seasonal variations, and human activities significantly impact nitrogen migration and transformation in agricultural watersheds. However, the issue of groundwater severely polluted by manure and sewage has received insufficient attention. To effectively control nitrogen pollution in agricultural watersheds, it is necessary to improve septic tanks and sewage networks, as well as implement scientific fertilization practices.