Abstract: The effect of cyflumetofen (CYF) on non-target organism earthworms (Eisenia fetida) in soil is unclear. In this study, a method was established for detection of CYF in earthworms. The samples were extracted with acetonitrile, the lipid in the earthworms was removed by liquid-liquid extraction, and then the concentration of CYF was detected by normal phase-HPLC. The results showed that the recoveries were 84%-88%, and the relative standard deviation was 1.3%-3.1% at the spiking levels of 0.5, 5, and 10 mg/kg, which verified the reliability of the method. According to the Biota-soil accumulation factor (BSAF), the enrichment concentration of the two enantiomers reached the maximum on the 7th day that the earthworms were enriched with CYF, and the enrichment ability of (+)-CYF in the earthworms was significantly higher than that of (−)-CYF. Long-term exposure of 0.5-10 mg/kg CYF for 28 days seriously inhibited the body weight of earthworms. The acute toxicity of cyflumetofen to earthworms was detected by filter paper method. The LC₅₀ of rac-CYF, (−)-CYF, and (+)-CYF to earthworms was 162.5, 323.1 and 309.4 mg/L, respectively. According to the combined toxicity analysis, AI = −0.03 was obtained. The simultaneously use of the two enantiomers of CYF will cause additive toxicity to earthworms.