Abstract: To elucidate the seasonal dynamics of dissolved organic matter (DOM) sources and their environmental driving mechanisms in an arid-region tributary, monthly water samples were collected from 15 cross-sections along the Kushui River (Ningxia, China) from March 2024 to February 2025. Water temperature (T), nutrients (TN, TP), and permanganate index (COD_Mn) were measured, and excitation–emission matrix (EEM) fluorescence spectra were obtained for DOM characterization. Fluorescent components were resolved using parallel factor analysis (PARAFAC), while fluorescence indices (FI, BIX, and HIX) were applied to infer DOM sources and humification characteristics. Canonical correspondence analysis (CCA) and random forest models were further employed to identify key environmental drivers. Five components were identified, including terrestrial humic-like (C1), microbial metabolite-like (C2), protein-like (C3), fulvic-like (C4), and tryptophan-like (C5) components. Spatial differences among river reaches were minor, whereas pronounced seasonal variability was observed. Biogenic components (C2 and C3) increased in summer, indicating enhanced autochthonous production and microbial reprocessing under elevated temperature. In contrast, terrestrial humic/fulvic components (C1 and C4) prevailed in autumn and winter, suggesting strengthened allochthonous inputs and intensified humification. Spring represented a transitional period from a terrestrial background towards enhanced biological production with mixed-source signals. FI (1.85-2.85) and BIX (0.85-1.35) generally supported a predominantly autochthonous DOM pool; however, a concurrent decrease in FI and BIX and an increase in HIX (0.53-0.80) in summer implied intensified terrestrial inputs and humification during runoff pulses. Both CCA and random forest consistently highlighted water temperature and total phosphorus as the strongest predictors, with COD_Mn and total nitrogen also contributing to variations in specific components. Collectively, these results demonstrate that DOM dynamics in the Kushui River are jointly regulated by temperature-driven biological processes and nutrient- and hydrology-mediated terrestrial inputs, leading to a seasonal shift from biogenic enhancement in summer to humic dominance in autumn and winter.