The present invention relates to a method and kit for isolating an extracellular vesicle (EV) from a biological sample using fractional precipitation. According to the method and kit, rapid isolation is possible, and an EV can be effectively isolated from various types of biological samples regardless of the sample sources. Unlike a method for isolating an EV on the basis of an existing precipitation method, the present invention can easily remove a salt by washing, and, thus, has the benefit of being able to isolate a highly pure EV with a high yield rate and can be applied to both small-scale EV isolation in research and diagnostic fields and large-scale EV isolation for industrial applications.