Neonicotinoids (NEOs) are the most widely used insecticides in the world. Nevertheless, the occurrence and distribution of NEOs in agricultural areas are not well understood. This study investigated the concentration, sources, ecological risks, and health risks of eight NEOs in the water of the Huai River, which flows through a typical agricultural area in China. The total concentration of NEOs in the river water ranged from 1.02 to 191.2 ng L⁻¹, with an average of 64.1 ng L⁻¹. Thiamethoxam was the dominant compound, with an average relative contribution of 42.5%. The average concentration of the total NEOs in downstream was significantly higher than that in upstream (p < 0.05). This may be related to the intensity of agricultural activities. The riverine NEO fluxes increased by approximately 12 times from the upper site to the lower site. More than 1.3 tons of NEOs in 2022 were transferred into Lake Hongze, the largest regulative lake on the Eastern Route of the South-to-North Water Diversion Project. Nonpoint sources were the major contributor to the total NEO inputs, and water use was the main output pathway. The risk assessment indicated that the individual NEOs in the river water presented low ecological risks. The NEO mixtures would produce chronic risks to aquatic invertebrates in 50% of the sampling sites, which were mostly distributed in downstream. Thus, more attention should be given to the downstream. Based on the Monte Carlo simulation, the health risks of NEOs via water consumption were estimated. The maximum chronic daily intakes were 8.4 × 10⁻⁴, 2.25 × 10⁻⁴, 1.27 × 10⁻⁴, 1.88 × 10⁻⁴ mg kg⁻¹ day⁻¹ for boys, girls, men, and women, respectively, which were approximately 2 orders of magnitude lower than the acceptable daily intake. Therefore, river water consumption would not be a concern for the public health.