Neonicotinoid pollution has increased rapidly and globally in recent years, posing significant risks to agricultural areas. Quantifying use and emission, transport and fate of these contaminants, and risks is critical for proper management of neonicotinoids in river basin. This study elucidates use and emissions of neonicotinoid pesticides in a typical large-scale agriculture basin of China, the Pearl River Basin, as well as the resulting agricultural non-point source pollution and related ecological risks using market surveys, data analysis, and the Soil and Water Assessment Tool. Neonicotinoid use in the basin was estimated at 1361 t in 2019, of which 83.1 % was used in agriculture. After application, approximately 99.1 t neonicotinoids were transported to the Pearl River, accounting for 7.2 % of the total applied. Estimated aquatic concentrations of neonicotinoids showed three seasonal peaks. Several distinct groups of neonicotinoid chemicals can be observed in the Pearl River, as estimated by the model. An estimated 3.9 % of the neonicotinoids used were transported to the South China Sea. Based on the present risk assessment result, several neonicotinoids posed risks to aquatic organism. Therefore, the use of alternative products and/or reduced use is deemed necessary. This study provides novel insights into the fate and ecological risks of neonicotinoid insecticides in large-scale watersheds, and underscores the need for greater efficiency of use and extensive environmental monitoring.