The components and sources of dissolved organic matter (DOM) in urban water bodies are extremely complicated due to multiple environmental and anthropogenic factors. The comprehensive analysis of the DOM molecular composition is crucial for deep understanding their reactivities, environmental behaviors, and fates in aquatic ecosystems, thereby improving our ability to monitor and protect aquatic systems. In this study, ultraviolet–visible spectroscopy (UV–Vis) and excitation-emission matrix fluorescence spectroscopy (EEM) were combined with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) to investigate the optical and molecular characteristics of DOM in the Yongding River (YDH) and Beiyun River (BYH), two typical urban rivers with distinct landscapes and anthropogenic influences in Beijing, China. Results showed that DOM in BYH was mainly composed of proteins from microbial and sewage sources with low aromaticity, molecular weight, and abundant saturated labile compounds, along with a significant presence of anthropogenic surfactants. Whereas the YDH was primarily composed of humic substances from plant- and soil-derived terrestrials, with higher aromaticity humification, unsaturation and refractory compounds. In addition, due to the accumulation of refractory compounds, the concentration of DOC and the degree of organic pollution increased from upstream to downstream of the rivers, manifested as an increase in humification and molecular weight at the molecular level. Our results confirmed that landscape urbanization broadly modified the structure of DOM to be more aliphatic and saturated. The findings of this study will provide important insights into the chemdiverse molecular composition of DOM undergoing different influences from human activities and provide significant guidance for water quality management in urban river ecosystems. Graphical Abstract