This study investigated the characteristics of dissolved organic matter (DOM) in the Huai River (Bengbu section) in China during wet and dry seasons. The methods included three-dimensional fluorescence spectroscopy (EEMs) combined with parallel factor analysis (PARAFAC) and self-organizing neural networks (SOM) to analyze DOM levels and composition. The results showed that the humus component (C1) of DOM was dominant in the water body, and there were significant seasonal differences. Rainfall and runoff significantly increased the input of humus organic matter in the wet season, and the fluorescence intensity and relative abundance of humic-like components (C1 and C2) were higher in the wet season than in the dry season. The concentration of plankton metabolites (protein-like component, C3) was higher in the dry season, reflecting increased microbial activity in the low water flow environment. Principal component analysis (PCA) revealed the dominant role of plankton metabolism and microbial activity on organic matter distribution, and the contribution of terrestrial organic matter to humus components through soil runoff. In summary, the seasonal variations in the Huai River water DOM were driven by both natural processes and human activities.