Abstract: Organic matter in lake water plays a key role in the global carbon cycle, especially in highly eutrophic water bodies, yet the linkage between its macroscopic distribution and molecular composition remains unclear. Taking Lake Qilu, a severely eutrophic enclosed plateau lake, as a case study, we employed a combined approach of physical particle size fractionation, three-dimensional fluorescence spectroscopy, and high-resolution mass spectrometry to analyze the particle size distribution, seasonal dynamics, and molecular composition of organic matter. The study revealed the transport patterns of organic matter from macroscopic distribution to molecular composition in Lake Qilu. The results showed that particulate organic matter dominated the lake water, with the highest concentration in the 100-250 μm size fraction, contributing 15.4% to the chemical oxygen demand (COD_Cr). During the dry season, organic matter larger than 10 μm prevailed, whereas during the wet season, the 0.05-5 μm fraction became dominant. Due to wind-induced resuspension of larger sediment particles during the dry season, the 10-250 μm fraction exhibited a high degree of humification. In contrast, the warm and rainy wet season triggered algal blooms, possibly enhancing the microbial source characteristics of dissolved organic matter (DOM) in the <0.05 μm fraction. PARAFAC analysis reveals that protein-like components dominate in dissolved organic matter (DOM). High-resolution mass spectrometry confirms that DOM smaller than 0.45 μm is primarily composed of lignin-like compounds, while DOM smaller than 0.05 μm has an increased proportion of sulfur-containing compounds compared to that larger than 0.05 μm. This is presumed to be caused by the large-scale mortality of submerged plants over a decade and the accumulation of agricultural non-point source pollution due to prolonged water exchange cycles. The organic matter composition in Lake Qilu exhibits particle size differentiation and seasonal dynamics, influenced by both terrigenous inputs and endogenous microbial activity, which are closely related to the lake’s enclosure, agricultural NPS pollution, and frequent algal blooms. Starting from particle size fractionation of organic matter in Lake Qilu, this study revealed its transport patterns from the ecosystem scale to the molecular level, providing a reference for understanding the carbon cycle in eutrophic plateau lakes.