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 (C 480 and C 400 ) and one protein-like component (C 310 ), with their relative importance following the order of C 480 ≥ C 400 > C 310 in the high-molecular-weight DOM (1 kDa-0.4 μm) and C 400 > C 480 > C 310 in 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 (SUVA 254 ). 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.