[Objectives]  Screening from cantharidin derivatives to find high efficient, low toxic and residual chemicals for R&D of new pesticides. [Methods]  Using bioassay and enzymatic determination methods to test 100 cantharidin derivatives. Stomach poison activity to the 3^rd-instar larvae of Mythimna separata was tested by using leaf disc feeding method and their activity of the detoxification enzyme system was determined by sublethal dose. [Results]  Bioassay results suggested that the effects of the derivatives Ⅰ-7^# and Ⅳ-13^# were better than norcantharidin; the stomach poison LC₅₀ of Ⅰ-7^# was similar to cantharidin and their LC₅₀ were 49.818 and 43.677,mg/mL respectively. The results of enzyme activity assay elucidated that cantharidin and its derivatives norcantharidin, Ⅰ-7^# and Ⅳ-13^# influenced detoxifying enzyme system of M. separata in different degrees. Among them, the specific activity of cytochrome P450 (P450) significantly reduced while carboxylesterase (CarE) is activated in different degrees by the cantharidin derivatives, and the specific activity of glutathione S-transferase (GST) generally showed activation at first and then inhibition. [Conclusion]  Cantharidin derivativesⅠ-7^# andⅣ-13^# have strong insecticidal activity against lepidopteran pests and are of value for development and utilization as biopesticides.