As one of the new environmental pollutants, neonicotinoid insecticides (NEOs) attract close attention due to their large dosage use and significant water solubility. NEOs are easily transferred into the aquatic environment with surface runoff and results in the threat to the water ecosystem and human health. The solid-phase extraction-ultra performance liquid chromatography-tandem mass spectrometry (SPE-HPLC-MS/MS) was used to analyse NEOs in Lake Poyang, aiming to reveal the spatial distribution characteristics of NEOs and analyse its pollution sources and ecological risks. The results showed that nine NEOs were detected to varying degrees in Lake Poyang Basin, among which dinotefuran (DIN), thiacloprid (THI), imidacloprid (IMI) and thiamethoxam (THIA)) with a detection rate of 100%. The concentration of the total NEOs ranged from 8.8 to 88.9 ng/L, with IMI the highest residues and the widest distribution (concentration range was 2.1-42.7 ng/L) which followed by THIA (concentration range is 0.7-18.3 ng/L). The main source of NEOs in Lake Poyang came from the non-point source input of agricultural activities. The ecological risk of NEOs to freshwater aquatic organisms in Lake Poyang Basin was evaluated based on species sensitivity distribution method and joint probability distribution curve method. The results showed that the order of ecological risk of the five insecticides was IMI> clothianidin (CLO)> THI> THIA> acetamiprid (ACE), and insects were the most sensitive species to NEOs. The joint probability distribution curve of NEOs showed that the adverse impact probability of IMI on 5% and 10% species was 28.55% and 0.3%, respectively. The acute risk of NEOs to aquatic organisms was not significant, while the chronic ecological risk of long-term NEOs application needed further attention.