〔Abstract〕 Objective To establish a stable and efficient method for the culture of mouse bone marrow mesenchymal stem cells and the extraction of exosomes. Methods The whole bone marrow, differential attachment and hypoxia environment were used to culture bone marrow mesenchymal stem cells(BM-MSCs).Then the cells were collected, the surface markers of stem cells were identified, and osteogenesis, adipogenesis and chondrogenesis were induced. The supernatant was collected, and the exosomes were extracted by overspeed separation and identified. Result The growth of mouse BMSCs was great, and the purity of the third generation cells could reach more than 95% and stable passage to about 15 generations. The cells expressed the markers of mesenchymal stem cells, and could be successfully induced to differentiate into osteogenesis,adipogenesis and chondrogenesis. The extracellular vesicles obtained by ultracentrifugation had typical exosome-like structure, and Western blot identification showed that they had characteristic protein markers of exosomes. Conclusion The whole bone marrow culture method makes the loss of BMSCs in mice minimum, and the differential attachment method under 5% oxygen environment makes the proliferation of BMSCs in mice fast and high purity. Moreover, the mouse BMSCs cultured by this method can secrete exosomes vesicles steadily. This provides a great method for the culture of mouse mesenchymal stem cells and the acquisition of their exosomes.