Methods of extraction and determination of tebuconazole enantiomers in earthworm (Eisenia fetida) were developed by capillary electrophoresis (CE) and high performance liquid chromatography (HPLC). Both CE and HPLC have excellent resolution and recovery. The linearity ranges were 2.9-102.4 mg/kg and 3.0-99.6 mg/kg for (+)-R-tebuconazole and (-)-S-tebuconazole respectively in CE, and from 0.56 to 1000 mg/kg for both enantiomers in HPLC. Enantioselective bioaccumulation in earthworms from soil was investigated under laboratory condition at concentrations of 10 and 50 mg/kg dw in soil. The uptake kinetics of (+)-R-tebuconazole fitted the first-order kinetics well with r2 0.97 and 0.94 under 10 and 50 mg/kg dw exposure condition, respectively, while (-)-S-tebuconazole with r2 0.75 and 0.22 did not show the same. Bioaccumulation of tebuconazole in earthworm tissues was enantioselective with a preferential accumulation of (+)-R-tebuconazole. The (+)-R-tebuconazole might also have biomagnifying effect potential in earthworm food chain with biota-sediment accumulation factor (BSAF) of 1.64 kg OC/kg lip in 10 mg/kg dw exposure group and 2.61 kg OC/kg lip in 50 mg/kg dw exposure group from soil to earthworm after 36 days. Although (-)-S-tebuconazole shares the same physicochemical properties with (+)-R-tebuconazole, it did not biomagnify. BSAFs of (-)-S-tebuconazole were 0.50 kg OC/kg lip (10 mg/kg dw tebuconazole exposure) and 0.28 kg OC/kg lip (50 mg/kg dw tebuconazole exposure) after 36 days, which was possibly owing to biotransformation or metabolism in earthworm tissues.