Stream order is considered a general predictor of composition of DOM in fluvial ecosystems, yet the longitudinal distributions and transformations of DOM can significantly vary depending on local or regional factors. However, DOM composition shifting along a river continuum and their interactions with stream metabolism are poorly understood. We collected surface water samples biweekly for nearly two years along a fluvial continuum monitored by the National Ecological Observatory Network (NEON), consisting of a small creek and two river sites. To characterize DOM composition, we employed the excitation and emission matrix fluorescence combined with parallel factor analysis (EEM-PARFAC), Fourier transform ion cyclotron resonance mass spectrometry (FI-ICR MS), and laboratory photochemical and microbial incubations. From upstream to downstream, dissolved organic carbon (DOC) concentrations increased. This increase is positively correlated with source-composition proxies that show DOM composition shifted from being dominated by aromatic, high-molecular-weight, allochthonous compounds at the upstream creek to being dominated by freshly produced, low-molecular-weight, autochthonous compounds in the two downstream river sites, suggesting the significance of riverine productivity and processing in altering DOM quantity and quality during the downstream transport. From upstream to downstream, DOM photodegradability did not show detectable changes, yet DOM became more recalcitrant to microbial degradation. These observations indicated the importance of humic compounds from riverine phytoplankton production and anthropogenic sources. Gross primary production (GPP) was higher at the downstream site, and it was correlated strongly with the DOM source shift to a higher contribution of autochthonous compounds. By contrast, ecosystem respiration did not differ significantly between the upstream creek and downstream river, and it cannot be explained by the quantity and quality of DOM. Overall, our findings highlighted the complexity of longitudinal distributions of DOM in relation to stream metabolism and that generalizations of DOM composition across stream orders should be considered with caution.