An environmentally-friendly and fast analytical method for the stereoselective determination of etoxazole was developed and then applied to estimate stereoselective bioaccumulation and elimination in zebrafish using SFC-MS/MS. Optimal enantioseparation conditions were determined using a Chiralpak IG-3 column and CO/MeOH mobile phase (80/20, v/v), at 3.0 mL/min within 1 min, 30°Me and 18 MPa. A modified QuEChERS method was developed for zebrafish sample pretreatment, and mean recoveries were 88.43-110.12% with relative standard deviations ranging from 0.32 to 5.34%. The enantioselectives of etoxazole enantiomers in zebrafish during uptake and depuration phases were evaluated. Significant enantioselective bioaccumulation was observed, with preferential accumulation of (-)-R-etoxazole compared to its antipode, during uptake at both low and high exposure concentrations. The toxic effects of etoxazole on zebrafish were further explored, and activities of antioxidant enzymes were determined in liver of zebrafish. Significant changes were observed in the SOD and GST activities and in the MDA levels, which indicated the occurrence of oxidative stress in liver of zebrafish. The toxic effects exhibited time- and dose-dependent properties. These results can facilitate the accurate risk evaluation of etoxazole and provide basic knowledge for further study of biotoxicity mechanisms.