Based on excitation emission matrix spectroscopy (EEMs) technology combined with the parallel factor analysis (PARAFAC), and UV-vis spectra, we analyzed the impact of seasonal variations on distribution and spectral characteristics of dissolved organic matter (DOM) in Zhoucun Reservoir. Results showed that the concentration of nitrogen and organic matter exhibited significant seasonal differences, and the reservoir has more total nitrogen and less total organic carbon in winter. The a₂₅₄ and a₃₅₅ in summer and autumn were both higher than those in winter and spring, which was consistent with the changes of organic matter. The E3/E4>3.5 showed that the fulvic acid accounted for the majority proportion in DOM; the concentrations of fulvic acid in summer and autumn were higher than those in winter and spring based on E2/E3. Moreover, the S_R>1 indicated the DOM exhibited autochthonous characteristics. One humic-like substances (C1), one fulvic-like substances (C2) and one protein-like substance (C3) were identified by PARAFAC model, and there exhibited significantly correlation coefficient in C1 & C2 & C3; Total fluorescence intensity and the fluorescence intensity of each component exhibited high concentration for summer and autumn and low concentration for winter and spring distribution characteristics. Total fluorescence intensity and the fluorescence intensity of each component exhibited significant seasonal difference. The DOM exhibited strong autochthonous component characteristics based on BIX and HIX indexes; Principal component analysis showed that the spectral characteristics exhibited obvious difference in the whole year, and the DOM spectral characteristics of summer and autumn are similar, and the DOM characteristics of water in winter and spring are similar. Moreover, the C1, C2 and C3 exhibited significantly correlation coefficient with DOM indices (FI, β/α) and dissolved organic carbon. Those three components and water quality (dissolved total nitrogen and dissolved organic carbon) of overlying water could be connected with spectral characteristic by nonlinear multiple. From all the results, it could contribute to explore and control the organic carbon pollution sources for the managers of Zhoucun Reservoir in the future.