Endogenous samples from the typical agricultural small watershed of Tumenxigou in Miyun Reservoir from 2022 to 2023 was collected to construct a fingerprint database. Six key sections of the river channel were collected in July and August for three-dimensional fluorescence spectroscopy determination, and the nitrogen sources of the small watershed were analyzed through parallel factor analysis. The results showed that the average mass concentration of nitrogen in the study area was 7.92 mg/L, and the main form was nitrate nitrogen. By combining the parallel factor analysis method with the excitation emission matrix, four fluorescent components were identified, mainly including two types of humus components (C1, C2) and protein like components (C3, C4). C1 is related to the application of agricultural organic fertilizers, C2 is related to composting, and C3 and C4 mainly come from rural domestic sewage or undecomposed urea fertilizers. The average fluorescence index of rural domestic sewage in the study area is about 2.16, and the average fluorescence index of soil leachate is about 1.83. Rural domestic sewage contributed about 35% to agricultural nonpoint sources in August. The fluctuation of water quality during the flood season in small watersheds is mainly related to the C1 and C2 components of humic substances. Therefore, attention should be paid to and control should be strengthened for the agricultural nonpoint sources brought by organic fertilizers in the study area.