Carbon fluxes in river networks represent a major component of the carbon cycle, but they are difficult to estimate at large scale. In particular, the physicochemical properties of organic matter (OM) and its contribution to river carbon fluxes remain poorly understood. In this context, this paper presents a dataset for quantifying and characterizing dissolved organic carbon (DOC) in a regional river basin, the Seine River basin in France (76 000 km<sup>2</sup>), which is subject to numerous human pressures. The dataset is the result of several sampling campaigns conducted over a 14-year period (2011–2024). A total of 1047 samples were collected from various water types (surface water, groundwater and treated effluents from wastewater treatment plants, WWTPs) at sites across the basin encompassing diverse land uses. Dissolved organic matter (DOM) was characterized both quantitatively, by measuring the concentration of dissolved organic carbon (DOC), and qualitatively, through analysis of its optical properties using UV-Visible absorbance and excitation–emission matrix (EEM) fluorescence spectroscopy. Additionally, using the 45-day incubation method, the biodegradable fraction of DOC, which plays a particularly significant role in river water quality, was estimated for 27 % of the samples. The content and properties of OM in the Seine River basin vary significantly depending on the site, measurement period, and type of water sampled. DOC concentrations are generally higher in WWTP discharges and gravel pits. The biodegradable fraction of DOC is higher in samples from WWTP discharges and from groundwater in a forested alluvial plain. OM in the basin generally displays a low level of aromaticity but is hydrophilic and characterized by strong biological activity. This biological activity is particularly pronounced in the treated effluents from small-capacity WWTPs, where OM is mainly of microbial origin, and in gravel pits, where OM consists primarily of protein-type compounds. Groundwater typically contains a mixture of OM of both terrestrial and microbial/biological origins. These data may be useful for future studies of (i) the organic carbon cycle at the regional basin scale, (ii) the characteristics of OM across different compartments of a hydrosystem, and (iii) river metabolism. The dataset is available at <a href="https://data.indores.fr/privateurl.xhtml?token=a6b58980-3280-4a1b-9311-a0b401955e75" target="_blank" rel="noopener">https://data.indores.fr/privateurl.xhtml?token=a6b58980-3280-4a1b-9311-a0b401955e75</a>.