The agricultural watershed of the Dianbu River is one of the main water sources of Chaohu Lake. It is very important to understand the aquatic ecosystem of Chaohu Lake to study the composition and source of dissolved organic matter (DOM) in this watershed. In combination with the fluorescence excitation-emission matrix spectroscopy of the watershed, the study made some relative processing with the parallel factor analysis (PARAFAC) method, including the Raman and Rayleigh scattering removing, component extraction and so on for the measured fluorescence spectrum matrix. So we realized to analyze the DOM of the agricultural watershed of Dianbu River, including the excitation-emission matrix fluorescence spectrum characteristics analysis, the fluorescence component ratio analysis and the correlation analysis between the fluorescence characteristics and water quality parameters and explored the DOM components and sources of the watershed. The results showed that the DOM of the agricultural watershed in the Dianbu River contained two effective fluorescence components, namely, one kind of protein (tryptophan-like component) and one kind of humus (fulcrum acid-like component). The proportion of fluorescence components indicated that the tryptophan-like component was the main component of the DOM in the watershed. The fluorescence index FI, the autogenous indicator BIX and humification indicator HIX indicated that the DOM has strong autogenous properties and weak humification characteristics. What’s more, its DOM of endogenous mainly comes from the metabolic activity of plants and other water microbial within the lotus pond, DOM of exogenous input from sewage and aquaculture feed. The endogenous of DOM made the main contribution to the organic matter source in the water; the DOC is positively correlated with the tryptophan-like component C1 of DOM, and the protein-like fluorescence component can be used for dynamic DOC tracking of this watershed. The pH value was positively correlated with the fulcruid-like component C2, so the pH value and the fulcruid-like component increased simultaneously, indicating that the water alkalization in this basin was accompanied by the increase of humus like substances in dissolved organic matter. Dissolved oxygen (DO) was negatively correlated with tryptophan-like component C1, indicating that the tryptophan-like component was affected by the content of dissolved oxygen dissolved oxygen content in water. This study traced the fluorescence characteristics of DOM and its component source response mechanism in the small agricultural watershed of Dianbu River, which could better understand its function in the ecosystem and its environmental geochemical cycle process, thus providing a certain scientific basis for the comprehensive environmental management of the watershed.