This invention describes a method and a device for efficient isolation of extracellular vesicles from animal and human biological fluids, as well as from culture fluid using equipment of standard diagnostic laboratories, that is, without the use of ultracentrifugation. These method and device can be applied for the diagnosis of various human diseases, as well as for therapeutic purposes, if the purified vesicles are used as an agent for drug delivery to the cells of the body. The device for the purification of extracellular vesicles contains at least two membrane filters: the first filter containing a membrane with pore sizes in the range from 400 to 600 nm, connected to the second filter containing a membrane with pores in the range from 95 to 200 nm. At the same time, the membranes of these filters are made of materials that practically do not bind biological polymers.