Human glutathione peroxidase1 (hGPx1) is a good antioxidant and potential drug, but the limited availability and poor stability of hGPx1 have affected its development and application. To solve this problem, we prepared a hGPx1 mutant (GPx1M) with high activity in an Escherichia coli BL21(DE3)cys auxotrophic strain using a single protein production (SPP) system. In this study, the GPx1M was conjugated with methoxypolyethylene glycol-succinimidyl succinate (SS-mPEG, Mw = 5 kDa) chains to enhance its stability. SS-mPEG-GPx1M and GPx1M exhibited similar enzymatic activity and stability toward pH and temperature change, and in a few cases, SS-mPEG-GPx1M was discovered to widen the range of pH stability and increase the temperature stability. Lys 38 was confirmed as PEGylated site by liquid-mass spectrometry. H9c2 cardiomyoblast cells and Sprague-Dawley (SD) rats were used to evaluate the effects of GPx1M and SS-mPEG-GPx1M on preventing or alleviating adriamycin (ADR)-mediated cardiotoxicity, respectively. The results indicated that GPx1M and SS-mPEG-GPx1M had good antioxidant effects in vitro and in vivo, and the effect of SS-mPEG-GPx1M is more prominent than GPx1M in vivo. Thus, PEGylation might be a promising method for the application of GPx1M as an important antioxidant and potential drug.