The invention discloses a preparation method of a silicon hydroxyl magnetic bead. The preparation method comprises the following steps that ferric trichloride, trisodium citrate, sodium acetate and polyhydric alcohol are mixed evenly to obtain a mixed solution, and Fe3O4 nanoparticles are prepared by a solvothermal method; and the surface of the Fe3O4 nanoparticles is coated with SiO2 films by atomic layer deposition to obtain the silicon hydroxyl magnetic bead. The Fe3O4 nanoparticles prepared by the solvothermal method are coated with the SiO2 films by atomic layer deposition (ALD), the SiO2films are plated on the surface of the Fe3O4 nanoparticles in the form of a monatomic film, the SiO2 films laminated by the method can grow at a constant speed on the surface and at defects of the Fe3O4 nanoparticles, the SiO2 films on the surface are a whole after coating, and the coating compactness of the SiO2 films on Fe3O4 is good and the SiO2 films are not prone to falling off, therefore, coating defects on the surface of the magnetic bead can be prevented from being generated. In addition, by adjusting the deposition conditions and the number of cycles, the thickness of the SiO2 filmscan be precisely controlled, the process is simple, operation can be standardized, no complicated operation is required, errors are small, and later screening of the magnetic bead is avoided, so thatthe purpose of improving the product quality and the stability of magnetic bead batches is achieved.