〔Abstract〕 Objective To explore method for isolating and culturing human epidermal stem cells(EPSCs)in vitroand isolating and purifying epidermal stem cell exsomes(EPSCs-Exo) by optimizing the technical process. Methods Firstly, the improved separating enzyme was used to isolate the EPSCs derived from human skin tissue. Then, an improved serum-free culture medium and 10 specific factors were combined to construct optimized 2D culture medium which could stimulate the growth of EPSCs, promote the secretion of EPSCs-Exo, maintain the stemness and proliferation of EPSCs, and delay the differentiation and maturation of EPSCs. Further, the conditions of differential centrifugation was optimized, and then the human EPSCs-Exo was successfully extracted with high efficiency and high purity. Results The human skin tissue was confirmed with the expressions of markers for epidermal cells. EPSCs were verified with high expression levels of integrin-α6, integrin-β1, P63 and CK19 by immunofluorescence staining and Western blot. The nanoparticle tracking analysis results showed the particles separated for EPSCs supernatant was saucepan with the detected diameter between 30-150 nm. The Western blot results showed the positive expression of membrane markers Tsg101, CD9 and CD63 and the negative expression of intracellular markers Calnexin and GAPDH. Conclusion The results show that the human-derived EPSCs have been successfully isolated and culturedin vitro, and the EPSCs-Exo have been successfully isolated and identified.