Abstract: A method for large-scale extraction of exosomes from fresh milk including: filtering fresh milk by using a hydrophilic and lipophobic nano-interfacial net membrane to remove cell fragments and fat particles in the fresh milk, and performing ultrafiltration by means of a tangential flow ultrafiltration device using an ultrafiltration membrane having a pore size of 100 kDa to 300 kDa to concentrate the clarified fresh milk; mixing the concentrated liquid with an aqueous two-phase solution prepared by combining polyethylene glycol and dextran, then performing phase separation, collecting an enriched exosome solution in the lower phase, and repeating one or two times to concentrate exosomes; further separating the concentrated exosomes by means of a column using a highly cross-linked agarose filler to remove other impurity proteins, and separating out exosomes and external vesicles having different diameters.