We measured nitrate in water (n = 4762) and nitrogen stable isotopes (d15N) (n = 353) of macroalgae and macroinvertebrates at different sites in a French karst river (Loue River) which has moderate nitrogen loading (* 23 kg nitrogen ha-1 - year-1). The main objective was to estimate whether nitrate in water and nitrogen stable isotopes of the biota could allow identification of the spatial and temporal variations in agriculture-related nitrogen losses to the river. Highest nitrate concentrations ([90% quantile) increased significantly over the last three decades but mean nitrate concentrations did not change significantly. Nitrate and biota d15N values tended to increase from upstream to downstream, although the d15N values decreased in the most downstream sites. Generalized additive mixed models allowed the identification of clear matching annual patterns of nitrate and biota d15N values characterized by recurrent autumn to winter maxima, supporting that agricultural nitrogen export and its assimilation within the river biota would mostly occur during the autumn and winter seasons. Overall, our results highlight the high vulnerability of karst river to nitrogen-related eutrophication.