The invention discloses application of entomopathogenic nematodes in the prevention and treatment of pest pupae of the family Noctuidae in the order Lepidoptera. A variety and line of entomopathogenicnematodes highly toxic to larvae of Spodoptera litura are acquired herein by screening; toxicity to pupae of Spodoptera litura is tested; experiments discover that when the density of nematodes increases to 100 IJ per pupa, corrected death rate of pupae treated with Steinernema carpocapsae Mex is up to 45.38%. Therefore, entomopathogenic nematodes can be applied to the prevention and treatment ofpupae of Spodoptera litura, and basis is provided for the application of entomopathogenic nematodes in the field prevention and treatment of Spodoptera litura at different development ages.