Abstract Magnetite nanoparticles were prepared by coprecipitation of Fe 2+ and Fe 3+ with NH 4OH, and then, silica was coated on the surface of the magnetite nanoparticles to form well-dispersed magnetic silica nanospheres. The size of spheres was controlled by changing the ratio of SiO 2 to Fe 3O 4. Amino–silane was covalently coupled to the surface of the magnetic silica nanospheres and activated by glutaraldehyde. The morphology and magnetic properties of magnetic silica nanospheres were examined by TEM and VSM. The nanospheres have an average size of 100–200 nm diameter and possess superparamagnetic characteristics. Powder X-ray diffraction measurements show the spinel structure for the magnetite particles coated by silica. By protein immobilization assay, it was proved that the amino–silane treated magnetic silica nanospheres could significantly improve the capacity of protein immobilization.