River systems represent important drivers of carbon loading, utilization and storage. However, underlying controls of hydrological transport and biological degradation on fluvial dissolved organic carbon (DOC) have yet to be revealed. Here, we explored spatiotemporal variability of DOC concentrations, components and sources, as well as its biodegradability in a headwater tributary of the Yangtze. We found that temporal rainfall stimulated terrestrial inputs and increased terrigenous DOC abundance. Hydrological transport was accompanied by biological generation and utilization of DOC, resulting in reduced labile components and accumulated recalcitrant components from tributaries to the main stem. Biodegradable DOC (BDOC) notably responded to temperature gradients over a 56-day laboratory incubation. Riverine DOC component, molecular weight and source highly predicted its biodegradation. Particularly, partial refractory (ultraviolet humic-like) fractions contributed to biological degradation of DOC, which was incompletely degraded from high-molecular to low-molecular weight compounds. The findings hope to supplement a new understanding of carbon fate under global change.