The Chishui River serves as a crucial tributary to the upper reaches of the Yangtze River. To enhance the protection of the water ecological environment in this area, it is essential to investigate the composition characteristics and sources of dissolved organic matter (DOM) within the upstream region. This study employed UET-visible absorption spectroscopy, three-dimensional fluorescence spectroscopy (EEMs), and parallel factor analysis (PARAFAC) to investigate the composition, distribution, and source of dissolved organic matter (DOM) in the Longjing small watershed, located in the headwater area of the Chishui River (Zhenxiong Section) in northeast Yunnan Province. The findings revealed that concentrations of DOC, CDOM, and FDOM gradually increased from top to bottom within the Longjing small watershed, with DOC concentrations ranging from 4.65 to 15.35 mg·L^-1, which is higher than those in other types of water bodies. Moreover, the humification degree and molecular weight level exhibited a pattern in which they decreased downstream, with lower reaches > middle reaches > upper reaches. DOM consisted of three fluorescent components: humus-like component C1, protein-like component C2, and long-wave humus component C3. Among these components, the tryptophan-based component C2 contributed significantly, accounting for 39.35% of the total fluorescence intensity, while C1 and C3 accounted for 32.17% and 28.48%, respectively.The origin of DOM primarily stemmed from autogenesis, characterized by weak humification; however, there was also some influence from both internal and external sources on certain sections within the Longjing small watershed due to comprehensive load effects.Under conditions such as isohigh reverse slope terrain, combined with nitrogen/phosphorus accumulation and vigorous microbial activity, DOM generation was promoted, altering its molecular structure.The C1 and C3 components exhibited a highly significant positive correlation, indicating their homology. TN and TP also demonstrated a significant positive correlation with the C1 and C3 components. Nitrogen and phosphorus in the Longjing small watershed predominantly existed in the form of organic nitrogen and organophosphorus, which combined with humus to contribute to exogenous DOM. This study provides valuable insights into the water quality of the Yangtze River's source waters, offering guidance for protecting its aquatic environment.