Spodoptera frugiperda is one of the most destructive crop pests in the world. Metarhizium rileyi is an entomopathogenic fungus specific for noctuid pests and is a very promising prospect in biological control against S. frugiperda. Two M. rileyi strains (XSBN200920 and HNQLZ200714) isolated from infected S. frugiperda were used to evaluate the virulence and biocontrol potential to different stages and instars of S. frugiperda. The results showed that XSBN200920 was significantly more virulent than HNQLZ200714 to eggs, larvae, pupae, and adults of S. frugiperda. In the larvae infected with the two M. rileyi strains, the activity of three protective enzymes (including peroxidase (POD), superoxide dismutase (SOD), catalase (CAT)) and two detoxifying enzymes (including glutathione-S transferase (GST) and carboxylesterase (CarE)) increased firstly and then decreased. The expression levels of protective enzymes and detoxification enzymes in larvae treated with XSBN200920 were greater than with HNQLZ200714. Furthermore, antioxidant stress-related gene (MrSOD and MrCAT family genes) expression in the two strains was measured by RT-qPCR (real-time quantitative PCR). The expression of these genes was significantly higher in the XSBN200920 strain compared to HNQLZ200714. There were also significant differences in the sensitivity of the two strains to the growth of different carbon and nitrogen sources and oxidative stress agents. In addition, the activity expression of antioxidant enzymes on the third day of culturing in XSBN200920 was significantly higher than with HNQLZ200714. In summary, the high virulence of M. rileyi XSBN200920 was not only determined by the expression levels of protective and detoxifying enzymes of the host but also regulated by the growth of entomogenic fungi and the resistance to the oxidative stress against S. frugiperda at different stages and instars. This study provides a theoretical fundament for the systematic control of Spodoptera frugiperda using Metarhizium rileyi.