Thiacloprid, a neonicotinoid insecticide, has been used widely in agriculture worldwide. In recent years, the adverse effects of neonicotinoid insecticides on non-target organisms have attracted more and more attention. In the present study, effects of thiacloprid on molecular biomarkers (GST, CarE, CAT, SOD, POD, and DNA damage) of earthworm Eisenia fetida were investigated using the artificial OECD soil for the first time. Earthworms were exposed to thiacloprid (1 and 3 mg/kg) for 7, 14, and 28 days and then transferred to the clean OECD soil for 35, 42, and 56 days. Results showed that activities of GST, CarE, CAT, SOD, and POD are inhibited following the exposure to thiacloprid at one or more sample times and then increased during the recovery course compared with the control. Significant DNA damage to E. fetida was also observed by olive tail moments in comet assay. These results suggested that thiacloprid could have harmful effect on earthworms, and these studied biomarkers might be used in the assessment of the risk of thiacloprid to the soil ecosystem environment.