Cold atmospheric plasma (CAP) is known as the versatile tool in different biological, and medical applications. In this study, we investigated the effect of cold plasma on diabetes via in vitro and in vivo assessments. We performed the in vitro assay to evaluate the impact of CAP on glycated glutathione peroxidase (GPx) through enzyme activity measurement as a function index and far- and near-UV circular dichroism (CD) and fluorescence analysis as structure indices. The result of in vitro assessment showed that the exposure of glycated GPx to plasma causes a considerable increase in enzyme activity up to 30%. Also, the evaluation of far- and near-UV CD and fluorescence analysis indicated a modification in the protein structure. According to obtained result from in vitro assessment, in vivo assay evaluated the effect of CAP on diabetic mice through analyzing of blood glucose level (BGL), advanced glycation end products (AGEs), antioxidant activity, oxidative stress biomarkers such as malondialdehyde (MDA), advanced oxidation protein products (AOPP), and oxidized low-density lipoprotein (oxLDL), and inflammation factors including tumor necrosis factor (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6). The result of in vivo experiment also showed a 20% increase in antioxidant activity. Also, the reduction in AGEs, oxidative stress biomarkers, and inflammatory cytokines concentrations was observed. The result of this study revealed that CAP could be useful in diabetes treatment and can be utilized as a complementary method for diabetes therapy.