The invention discloses a hydroxyapatite-based method for separating and purifying various exosome subgroups. The method comprises the following steps: firstly, separating and purifying a total exosome suspension from a medium or a biological fluid sample; and then, on the basis of hydroxyapatite, implementing chromatographic separation, so that the various exosome subgroups are obtained, wherein exactly, the step comprises the following operations: mixing the total exosome suspension with the hydroxyapatite, implementing incubation under a shaking condition, transferring an incubated material to an affinity chromatographic column, washing the column by virtue of a low-concentration sodium phosphate solution, then sequentially eluting the column by virtue of sodium phosphate solutions of which concentrations are gradually increased, so that various eluents are obtained, and removing impurities, so that the various exosome subgroups are obtained. The method is simple and rapid; the obtained various exosome subgroups can nearly avoid overlap in distribution of protein molecules, RNA molecules, DNA molecules and the like; moreover, an exosome desorption process is mild and obtained exosome can be further applied; and the method can be also used for specifically enriching exosome excreted by cancer cells. The method has a good clinical application foundation; and the method is great in application prospect.