Seasonal fluctuations present significant challenges to drinking water treatment by altering the properties of Dissolved Organic Matter (DOM) within watersheds, thereby influencing the potential for disinfection by-product (DBP) formation. DOM is a complex mixture of organic matter that serves as a critical DBP precursor and is closely linked to adverse health outcomes. The prediction of DBP formation is complicated by the variability in DOM concentrations and compositions in lake source water, a situation exacerbated by seasonal changes in water systems. We examined the seasonality of lake DBP formation potential (DBPFP) and the dynamics of precursors across four distinct seasons based on water temperature. Utilizing the Excitation-Emission Matrix (EEM) coupled with parallel factorial (PARAFAC) analysis, three-dimensional fluorescence difference spectroscopy (3D-FDS), and molecular weight distribution (MWD), we elucidated the compositions and fates of lake DBP precursors. The findings revealed that DBPFP (THMFP and HAAFP) were markedly influenced by seasonal variations, with peak fluorescence intensity occurring during the summer. Contributions to the water system were dominated by microbial metabolites (region IV) and protein-like substances (region I and region II). 3D-FDS analysis further substantiated the low homogeneity of DBP precursors between summer and autumn, with fulvic acid (FA) substances comprising up to 36.89 % of the variance. Distinct fluorescence intensities were detected at Peak B (266.29 A.U.) and Peak T (376.19 A.U.). Throughout the year, a total of four fluorescent components were characterized, encompassing humic-like substances (C3) and protein-like substances (C1, C2, C4), indicative of biogenic pollution. The source of DBP precursors was identified as small molecular weight organic matter (0.2–5 KDa), resulting from microbial metabolic processes and the degradation of aquatic plants. In addition, external factors such as chlorination, pH levels, and contact time significantly influence THMFP and HAAFP. Overall, these findings advance our comprehension of the transport and fate of DBP precursors within drinking water sources and lake ecosystems. This knowledge is pivotal for optimizing water treatment protocols in relevant water treatment facilities. © 2024 Elsevier B.V.