Neonicotinoid pesticides (NNs) are increasingly used in agriculture, which may pose significant threats to aquatic organisms in receiving rivers. However, no studies have explored their entire process from application and transport to receptors within river basins. Here, we estimated the usage and loss of NNs in the Dongting Lake Basin in China using modeling approaches, and assessed NNs-associated aquatic ecological risks. Our research data showed that the annual usage of the nine NNs reached 1895 tonnes in the basin, with the peri-urban areas being the major users. We further calibrated and validated a SWAT model using various 13-years hydrological data and field measured NNs concentration data. The simulated total annual loss of NNs was 121 tonnes in the entire basin, 94 tonnes of which were discharged into the Dongting Lake. An obvious monthly variation was observed in the lake basin, with relatively higher NNs concentrations being found in summer. Results from the ecological risk assessment showed that NNs posed significant risks to aquatic organisms in approximately 11.2 % of river sections in the whole basin. The present study underscores the significant issue of NNs loss in the Dongting Lake Basin and warrants great attention to their potential risks to aquatic organisms.