A simple and efficient method for separating migrasomes from macrophages is provided. The method separates migrasomes having a diameter range of 0.5 micrometers (μm) to 3 μm by intercepting through a filter and eluting through reverse filtration successfully. The separated migrasome has a vesicle-liked structure and wrinkles on its surface, and the separated migrasome has a diameter over 500 nanometers (nm). The separated migrasomes express their characteristic proteins PIGK, EOGT, and TSPAN4, but do not express specific markers TSG101 and ALIX of EVs, indicating that the separated migrasomes are a unique type of vesicles distinct from extracellular vesicles (EVs). The integrity of ribonucleic acids (RNA) carried by the migrasomes is not affected. The method for separating migrasomes from macrophages has the characteristics of simplicity, high efficiency, good controllability, good repeatability, and low cost, and large special equipment is not needed.