Objective To investigate the isolation and identification of extracellular vesicles (EVs) from rat bone marrow mesenchymal stem cells (BMSCs) and the effect of -80DegreesCelsius cryopreservation on the structural integrity of the EVs' membrane. Methods EVs were isolated and purified from the culture supernatant of bone marrow mesenchymal stem cells (BMSC-CS) by ultrahigh velocity centrifugation. The size and morphology of EVs were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Western blotting was performed to detect the expression of CD63 protein. The integrity of the EVs' membrane structure was observed by TEM at different time after storage at -80DegreesCelsius. Results SEM showed that a large number of EVs with membrane structures, round or oval and ranged in size from 50 to 1000 nm were attached onto BMSC surface. Under TEM, BMSC-EVs were similar in morphology and size to them under SEM. Western blotting revealed that BMSC-EV expressed the marker protein CD63. BMSC-EVs had a complete membrane structure at the first month and the third month, whereas BMSC-EV's membrane structure ruptured at the sixth month after storage at -80DegreesCelsius. Conclusion BMSC-EVs is successfully extracted, and the membrane structure integrity get worse with the prolongation of cryopreservation time.