Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown great promise for regeneration and immunomodulation. However, efficient and scalable methods for their preparation are still lacking. In this study, we present the adoption of a label-free technique known as "EXODUS" to isolate and purify MSC-EVs from the conditioned medium. Our findings indicate that EXODUS can rapidly isolate EVs from 10 mL of conditioned medium with a 5-fold higher yield compared to conventional approaches, including ultracentrifugation (UC) and polyethylene glycol precipitation (PEG) methods. Additionally, pre-storing the conditioned medium at 4°C for 1 week resulted in a ~2-fold higher yield of MSC-EVs compared to the freshly prepared medium. However, storing the purified EV particles at 4°C for 1 month led to a 2-fold reduction in particle concentration. Furthermore, we found that MSC-EVs isolated using EXODUS exhibit higher expression levels of EV markers such as Alix, Flotillin1, CD81, and TSG101 in comparison to PEG and UC methods. We also discovered that MSC-EVs isolated using EXODUS are enriched in response to cytokine, collagen-containing extracellular matrix, and calcium ion binding compared to PEG method and enriched in extracellular structure organization, extracellular matrix, and extracellular matrix structure constituents compared to UC. Finally, we demonstrated that MSC-EVs isolated using EXODUS exhibit greater potential in animal organ development, tissue development, and anatomical structure morphogenesis compared to the UC. These findings suggest that EXODUS is a suitable method for the large-scale preparation of high-quality MSC-EVs for various clinical applications.