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Arctic rivers are sensitive to climate and environmental change, but the biogeochemical response remains poorly understood. Monthly size‐fractionated dissolved organic matter (DOM) samples from the lower Yukon River were characterized using UV–visible, fluorescence, and Fourier transform‐infrared (FT‐IR) spectroscopy techniques. The EEM‐PARAFAC analysis revealed three major fluorescent DOM components, including two humic‐like components (C₄₈₀and C₄₀₀) and one protein‐like component (C₃₁₀), with their relative importance following the order of C₄₈₀ ≥ C₄₀₀ > C₃₁₀in the high‐molecular‐weight DOM (1 kDa–0.4 μm) and C₄₀₀ > C₄₈₀ > C₃₁₀in the low‐molecular‐weight DOM pool (< 1 kDa). Transformation in DOM and change in sources were manifested in major fluorescent components and optical properties, including biological index (BIX), humification index (HIX), spectral slope (S_275–295) and specific UV absorbance at 254 nm (SUVA₂₅₄). These changes occurred within different DOM size‐fractions and among ice‐covered, spring freshet, and open seasons. Joint analysis of EEM and FT‐IR spectra using a data fusion technique showed that humic‐like DOM is mostly associated with C─H, C═C, and C─O bonds, while protein‐like DOM is correlated more with C─N and N─H related structures. DOM aromaticity and the ratios of HIX to BIX and protein‐like to humic‐like components may be used as a compelling proxy to measure change in source waters and to infer permafrost dynamics. Our results provide insight into the seasonal variation in DOM composition for different size‐fractions in the lower Yukon River, and a baseline dataset against which future changes can be understood in the context of arctic basin biogeochemical cycling.