Method summary This innovative method can successfully isolate extracellular vesicles from tissues based on frozen section pretreatment to obtain tissue fragments, followed by enzymatic hydrolysis, differential ultracentrifugation, size exclusion chromatography and ultrafiltration procedures. Compared with the tissue extracellular vesicle isolation methods based on the scalpel section, the new method is more specimen-saving, repeatable, standardized, time-saving, and easier to be magnified and automated. Extracellular vesicles (EVs) are small vesicles mediating intercellular communications that have been widely used in disease diagnosis. Extracting EVs from tissues is of great importance, but current approaches are finite and the EV yield is limited. Here, the authors introduced a new method to increase EV yield based on frozen sectioning. With a standardized, semiautomated tissue-slicing procedure in a cryostat, the authors successfully isolated EVs from hearts, kidneys and stomachs. The morphology, size distribution and purity of those isolated EVs were evaluated. Additionally, compared with the traditional scalpel section method, they confirmed the higher yield of tissue-derived EVs with the cryostat-based method. The authors believe that the new method they developed would largely facilitate the research and clinical application of EVs.