The invention provides carboxyl magnetic microspheres, a preparation method and an application. The surfaces of mesoporous polymer microspheres are modified with functional groups with a physical adsorption method, carboxyl magnetic microspheres are prepared with an in-situ coprecipitation synthesis method, and magnetic microspheres modified with carboxyl repeatedly is prepared by silica coating,amino modification and carboxyl re-modification. The carboxyl repeatedly modified magnetic microspheres are functional composite microspheres, have the advantages of magnetic responsiveness, high stability, better biological compatibility, uniform particle size, good monodispersity, controllable magnetic content and good batch repeatability, are suitable for large-scale stable preparation in industry, and can be widely applied to the fields of cell separation, immune detection, protein purification, immobilized enzyme, environmental protection and the like.