Extracellular vesicles (EVs) are nanosized particles secreted by cells, reflecting the characteristics of their mother cells. Mesenchymal stem cell (MSC)-derived EVs show potential for regenerative wound healing and modulating immune responses. However, traditional isolation methods suffer from limited yield and purity. This study reports a two-step tangential flow filtration (TFF) system utilizing track-etched membranes with uniform cylindrical nanopores (50 and 200 nm) to isolate small EVs (sEVs) via a size-exclusion mechanism. Comparative analysis reveals that this nanoporous membrane-based TFF (Nano-TFF) system achieves a separation efficiency exceeding twofold and a purity surpassing 90% over existing ultrafiltration systems. The efficacy of these sEVs was validated in a wound animal model, where sEV-loaded dressings enhanced recovery. The Nano-TFF system provides precise, high-efficiency separation for various bioactive agents requiring high-purity isolation