Lakes in cold and arid regions represent vital global freshwater resources, yet the partitioning mechanisms of dissolved organic matter (DOM) during freeze–thaw cycles and associated microbial–environmental drivers remain poorly understood under extreme low-temperature and aridity stresses. This study investigates four characteristic lakes in Inner Mongolia, China, employing ultraviolet-visible spectroscopy, three-dimensional fluorescence spectroscopy, and 16S rRNA sequencing to analyze DOM optical properties, microbial communities, composition, sources, and multi-scale partitioning behavior across ice–water phases. The key results show the following: (1) DOM in lake waters exhibited mixed autochthonous and allochthonous origins with low humification but significant lake-specific variability, whereas ice DOM was predominantly autochthonous with lower humification and showed vertical fractionation—increasing the molecular weight and humification with depth—indicating selective partitioning during freezing; (2) salinity, pH, water depth, and ice thickness were key factors shaping DOM-related microbial community structure, with microorganisms significantly promoting the transformation of protein-like components in ice; (3) DOM migration and transformation displayed clear salinity-driven vertical stratification during freeze–thaw processes, with the freezing rate and salinity being the primary controlling factors. These findings elucidate DOM interfacial partitioning mechanisms and ecological impacts in cold-arid lakes, providing critical theoretical and empirical support for assessing carbon cycling and ecosystem risks under climate change and intensifying salinization, and offering new insights for conservation and management of vulnerable icy lake ecosystems.