This study investigates two typical urban rivers in Wuhan—Xunsi River, which receives substantial anthropogenic pollution, and Qingshangang River, which has undergone ecological restoration. By comparing the concentration, optical properties, and fluorescence characteristics of dissolved organic matter (DOM), we systematically elucidate the mechanisms through which human activities influence DOM composition, sources, and dynamic changes. The results show that anthropogenic pollution input is the primary driver of spatial differences in DOM. Xunsi River exhibited significantly higher DOC concentrations, greater aromaticity, and elevated levels of all fluorescent components compared to Qingshangang River. Source apportionment indicated that DOM in Xunsi River is dominated by internal inputs and is enriched in labile protein-like substances, whereas DOM in Qingshangang River is a mixture of autochthonous and allochthonous sources, with more stable humic-like substances prevailing. Temporally, DOM characteristics in Xunsi River showed pronounced seasonal variations due to strong anthropogenic disturbances; in particular, protein-like components increased markedly during the wet season under the influence of sewage–stormwater mixing. In contrast, DOM composition in Qingshangang River remained stable across seasons due to controlled external inputs and the predominance of humic-like substances. This study demonstrates that urban sewage discharge not only directly elevates organic loading in rivers but also indirectly modulates internal metabolism by altering nutrient levels, jointly shaping the characteristics of DOM. Therefore, the strategy of controlling exogenous pollution in coordination with endogenous regulation can provide a scientific basis for improving river water quality and optimizing regional carbon cycle management.