Abstract: Disclosed is a method of isolating extracellular vesicles using an aqueous two-phase system (ATPS), including (a) preparing an ATPS by mixing a first material and a second material, which are immiscible with each other, with a body fluid or an aqueous solution containing extracellular vesicles and (b) isolating extracellular vesicles concentrated in the second material of the ATPS. This method can exhibit very high isolation efficiency, a simple isolation manner, and a very short isolation time. The isolation of extracellular vesicles using the ATPS requires no ultracentrifuge and achieves almost 100% isolation efficiency within a short time of about 10˜20 min, and thus the method of the invention is practical, is economical due to low costs thereof, can increase the purity of extracellular vesicles contaminated with protein, enables the diagnosis of disease using the isolated extracellular vesicles, and can be applied to various fields using extracellular vesicles.