Glutathione peroxidase 1 (GPx1) is an important antioxidant selenium enzyme and has a good prospect for drug development. However, the expression of GPx1 requires a complex expression mechanism, which makes the drug development of recombinant GPx1 (rGPx1) difficult. In the previous study, we expressed highly active rhGPx1 in amber-less Escherichia coli by using a novel chimeric tRNA^UTuT6. However, the antioxidant effect of rhGPx1 at the cellular and animal levels has not been verified. In this study, we established isoproterenol (ISO)-induced oxidative stress injury models to study the antioxidant effect of rhGPx1 at the cellular and animal levels. Meanwhile, in order to more accurately reflect the antioxidant effect of rGPx1 in mice, we used the same method to express recombinant mouse GPx1 (rmGPx1) as a control for rhGPx1. The results of a study showed that rhGPx1 has a good antioxidant effect at the cellular and animal levels. However, due to species differences, rhGPx1 had immunogenicity in mice and antibodies of rhGPx1 could inhibit its antioxidant activity, so the antioxidant effect of rhGPx1 was not as good as rmGPx1 in mice. Nevertheless, this study provides a reliable theoretical basis for the development of rhGPx1 as an antioxidant drug.