Objective To compare the differences and characteristics of extracellular vesicles in different tissues extracted by different methods, and to explore the separation and purification methods of extracellular vesicles in tissues, so as to lay a foundation for studying the function and mechanism of extracellular vesicles in tissue microenvironment. Methods Collagenase digestion was performed on four tissues:kidney, prostate, skin and stomach, to obtain decellularized tissues and to prepare extracellular matrix suspensions. Qiagen exosome extraction kit and SBI exosome extraction kit and ultracentrifugation (UC) method were used to extract the extracellular vesicles. The ATS particle size analyzer qNano technique was used to measure the particle size and concentration of the extracellular vesicles. Results Purified extracellular vesicles were obtained by the three methods from the extracellular matrix suspensions of the four tissues, and the main vesicle type was exosomes. The distribution of particle size was different among the different types of tissues, but the difference of particle size was not significant (P>0.05). In homogeneous tissues, the extracellular vesicles obtained by UC method had the highest concentration, which were significantly different compared with the concentrations of the extracellular vesicles obtained by the other two methods (P<0.01). The concentrations of the extracellular vesicles from prostate and kidney were higher, and those from stomach and skin were lower, and the difference was statistically significant (P<0.05). Conclusion The concentration of extracellular vesicles extracted by UC method is the highest in the various types of tissue microenvironments. There is a significant difference in the concentrations of extracellular vesicles from different types of tissue microenvironments.