In view of the significance of food safety and the possible relationship between residual enrichment and acute toxicity for pesticides in different aquatic organisms, it is essential to establish a sensitive and reliable determination method for pesticides in different aquatic organisms to analyze the enrichment levels. Quinone outside inhibitor fungicides (QoIs) are lipophilic fungicides that pose environmental threats to aquatic organisms. Previous research has mainly focused on QoI residues in aquatic organisms under chronic toxicity, whereas less is known about how pesticide residues differ among aquatic organism under acutely toxic conditions. In the present study, the residues of QoIs in aquatic organisms (Danio rerio, Rana pipiens, Cherax quadricarinatus, Misgurnus anguillicaudatus, Corbicula fluminea, and Ampullaria gigas) were analyzed by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) coupled with a proposed QuEChERS method. The proposed method was validated in terms of linearity (coefficients of determination of 0.9980-0.9999), the limits of quantification (0.01μg·kg-1), the relative standard deviation (0.6-4.4%), and recovery (70.12-118.15%). The results demonstrated that the proposed method fulfilled the requirements for pesticide analysis in all tested aquatic organisms. The residues of QoIs in the same aquatic organism exposed to QoI concentrations of 5 and 500μg L-1 decreased in the order pyraoxystrobin > pyraclostrobin > triclopyricarb > picoxystrobin > azoxystrobin > fluoxastrobin. Furthermore, the acute toxicity was strongly correlated with the enrichment level of the QoIs in aquatic organisms. This study provides the first documentation of a correlation between the enrichment level of QoIs and acute toxicity in aquatic organisms, which provides a basis for the management of agrochemicals considering aquatic ecological risks.