Due to their effectiveness at low doses and relative safety for non-target species, plant essential oils (EOs) are considered ideal alternatives to conventional pesticides for pest control. In this study, the chemical composition of Thymus serpyllum (T. serpyllum) EO was construed by Gas Chromatography-Mass Spectrometry (GC-MS), and its larvicidal and ovicidal activity against omnivorous pests Spodoptera litura (S. litura) was assessed. The effects of T. serpyllum EO on the activities of antioxidant detoxification enzymes were also measured. GC–MS analysis revealed that the main constituents of T. serpyllum EO were thymol (42.1%), p-cymene (22.4%), and γ-terpinene (18.6%). In the larvicidal toxicity experiment, the T. serpyllum EO demonstrated LC50 values of 0.606 and 0.664 mg/mL against the second- and third-instar larvae of S. litura, respectively, after 48 h exposure. Moreover, an EC50 value of 0.905 mg/mL was measured against S. litura eggs. In S. litura, T. serpyllum EO treatment reduced the enzymatic activity of ESTs and GST and, conversely, increased the enzymatic activity of AChE. Overall, this study demonstrated that T. serpyllum EO has the potential to be implemented as a novel eco-friendly insecticide against S. litura.