Under the combined influence of various factors, the dissolved organic matter (DOM) in the drainage basin presents complex variation characteristics. In this study, we investigated the spectral characteristics of DOM in the middle reach of the Huai River aquatic systems under the influence of complex conditions (i.e., agricultural nonpoint pollution, coal mining activities, photovoltaics on the water surface, and rural sewage). In addition to the general water quality indices, ultraviolet-visible (UV-Vis) absorption spectroscopy, the excitation-emission matrix spectroscopy (EEMs) combined with the parallel factor analysis (PARAFAC), the fluorescence regional integration (FRI), and the fluorescence indices were used to describe the spectral characteristics of DOM. The results show that agricultural non-point source input from agricultural activities deteriorates water quality, but the coal mining activities weaken the agricultural non-point pollution intensity (i.e., occupy farmland, and subsidence area collected agricultural runoff). The PARAFAC model was composed of the following three chemical components: fulvic-like (C1), humic-like (C2), and tryptophan-like (C3). The results of PRAFAC and FRI indicated that the photovoltaic industry significantly influenced the properties of DOM in the aquatic systems by slowing the photochemical reactions. The principal component analysis (PCA) and Adonis test showed that under different influencing conditions, the aquatic systems within the four study areas showed significant differences. Spectral characteristics of DOM can sensitively indicate water distinction. It is of practical significance to monitor the change characteristics of DOM in a minor watershed under complex influencing factors by spectroscopy.