The sources and composition of dissolved organic matter (DOM) in rivers are critical to water quality and aquatic ecosystems. Studies on detailed composition of organic matter in rivers in the farming-pastoral ecotone are relatively limited in the research community. To better understand the characteristics and dynamics of DOM, Yang River in North China was selected as the study area because of its profound influences on the farming-pastoral ecotone nearby. A combination of fluorescence spectroscopy and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) techniques revealed that the DOM composition of Yang River is driven by land use. DOM in Yang River is predominantly imported from allochthonous inputs, together with agricultural runoff, pastureland, and urban sewage, causing a comprehensive impact on DOM. In detail, DOM associated with cropland inputs was dominated by lignin-like species, with higher nitrogen content. In comparison, DOM related to grassland is more diverse and susceptible to degradation. An increase in urban areas led to an increase in sulfur-containing compounds, while their oxygen, nitrogen, and aromaticity contents were significantly lower than those in cropland. Interestingly, urban-influenced lignin-like compounds may be associated with the effluents from the pulp and paper mill. Additionally, synthetic surfactants from the lower section of the river were also structurally identified by tandem mass spectrometry. Overall, this study could provide valuable insights into the DOM sources and their transformation dynamics at a molecular level, which could be an indicator for riverine water quality management and be applied to other farming-pastoral ecotones straightforward.