AbstractUrbanization and agricultural land use can increase the transport of nutrients to fluvial ecosystems; yet, the overall impact of urban and nonpoint agricultural inputs on the composition, bio-lability, and the fate of fluvial dissolved organic matter (DOM) remains poorly understood. We investigated the optical and molecular composition and bio-lability of DOM in 76 streams and rivers with varying sizes of catchment area (1 ~ 4850 km²) along large gradients of urban and agricultural land use. Optical spectroscopy and Fourier transform ion cyclotron mass spectrometry (FT-ICR MS) were used to determine the chemical composition of DOM, and laboratory bio-incubations were used to assess DOM bio-lability. We found that urban and agricultural land use intensification was associated with the reduced relative contribution of DOM from pristine soils, enriched dissolved organic carbon (DOC), and higher contributions of aliphatic and peptide-like DOM, as well as enrichment of N- and S-containing molecular formulae. Specifically, FT-ICR MS revealed that the relative abundance of CHO-containing formulae decreased while that of CHOS-containing formulae increased with increasing urban land use. DOM bio-availability was higher in rivers draining urbanized and agricultural landscapes and was related to decreased DOM aromaticity and increased proportions of energy-rich aliphatic formulae. Our results suggested that DOM from urbanized and agricultural catchments significantly altered the chemical composition and bio-availability of DOM with potential effects on ecosystem metabolism and the fate of organic carbon in the downstream-linked streams and rivers. Anthropogenic disturbances may thus boost the microbial processing of organic carbon, including uptake, retention, and outgassing in the impacted ecosystems. Graphical Abstract