The invention discloses a silicon dioxide nano magnetic microsphere. The particle size is 50-600 nm, an inner core is a magnetic nanoparticle with the particle size of 10-500 nm, compact silicon dioxide with the thickness of 1-10 nm is arranged in a middle layer, mesoporous silicon dioxide is arranged in an outer layer, the specific surface area of the nano magnetic microsphere is 10-500 m<2> / g, and the average aperture is 2-50 nm. A preparation method for the silicon dioxide nano magnetic microsphere comprises the steps as follows: dispersing the magnetic nanoparticle in a solvent, adding a compact silicon dioxide precursor, stirring for reaction, and coating the outer surface of the magnetic nanoparticle with a layer of compact silicon dioxide; adding a mesoporous silicon dioxide precursor and stirring for reaction; and separating and carrying out mesoporous formation to obtain the silicon dioxide nano magnetic microsphere with a mesoporous structure. The silicon dioxide nano magnetic microsphere has the functions of large specific surface area, adjustable aperture and capability of loading molecules with different sizes, and is widely applied to the aspects of cell separation, enzyme immobilization, protein separation, immunodetection, immunodiagnosis of water body pollutants and the like.