Mesenchymal stem cells (MSCs) derived exosomes, as a cell-free therapy to replace MSCs, have higher safety and great potential in regenerative medicine. The isolation of exosomes is a challenge that complicates their application. The commonly used ultracentrifugation and tangential flow filtration methods are inconvenient due to the need for expensive instruments and ultrafilter membranes. The cost of commercial kits limits their application in handling large samples. PEG precipitation is convenient and cost-effective. However, there is a need for optimized and standardized isolation methods based on PEG precipitation in different cell types or liquids to ensure consistent quality and yield. In this work, we optimized the PEG precipitation method for exosomes isolation and compared its effectiveness to two commonly used methods: ultracentrifugation (UC) and commercial exosome isolation kits (ExoQuick). The recovery rate of the optimized PEG method ( about 61.74%) was comparable to that of the commercial ExoQuick kit (about 62.19%), which was significantly higher than UC (about 45.80%). Exosome cargo analysis validated no significant differences in miRNA and protein profiles associated with the proliferation and migration of exosomes isolated by UC and PEG precipitation, which was confirmed by gap closure and CCK8 assays. These findings suggest that PEG-based exosomes isolation could be a highly efficient and high-quality method and may facilitate the development of exosome-based therapies for regenerative medicine.