The invention discloses a method for preparing magnetic composite nanoparticles with a core-shell structure. In the method, electrostatic self-assembly and seeded emulsion polymerization are combined, wherein the electrostatic self-assembly is used to synthesize particles with the core-shell structure first and then emulsion polymerization is performed by using the particles with the core-shell structure seeds and using an initiator to prepare the magnetic composite nanoparticles with the sandwich core-shell structure which has three or more layers. The magnetic composite particles prepared by the method have a multi-layer sandwich core-shell structure, wherein in the structure, the core is a polymer microsphere, a Fe3O4 magnetic particle layer is in the middle, and different polymer layers are covered outside. The prepared particles have stable structures, strong and uniformly-distributed magnetism, smooth surfaces, controllable sizes, high stability and low cost, the thicknesses of the covering layers of the prepared particles can be controlled by nanoscale, and the adaptability of the prepared particles is high. Particles with specific surface groups can be prepared according to requirements and can be used in fields of biochemical separation, targeting preparation, immobilized enzyme, immunoassay, catalysis study and the like.