Abstract: This study investigates the particle size distribution and seasonal dynamics of organic matter in the heavily eutrophic Lake Qilu, utilizing physical fractionation, spectral techniques, and mass spectrometry. The results indicate that organic matter is predominantly particulate, with the highest concentrations found in the 100-200 μm size fraction. The dry season is characterized by the dominance of large-sized particulate organic matter, while a shift towards smaller-sized components occurs during the wet season. Due to wind-induced sediment resuspension in the dry season, large-sized dissolved organic matter exhibits a higher degree of humification. In contrast, the concurrent high temperatures and rainfall during the wet season promote algal blooms, resulting in small-sized dissolved organic matter with distinct microbial signatures. Parallel factor analysis of fluorescence spectra revealed a predominance of protein-like components, and Fourier transform ion cyclotron resonance mass spectrometry confirmed that lignin-like compounds are the most abundant, with an increase in sulfur-containing compounds within the smaller size fractions. In conclusion, the composition of organic matter demonstrates significant size fractionation and seasonal dynamics, influenced by both allochthonous input and autochthonous microbial activities. These characteristics are closely associated with the lake's closed nature, agricultural pollution, and high productivity, providing new insights for understanding the carbon cycle in eutrophic plateau lakes.