The separation process is the main stage of recombinant production. With the advancement of nanotechnology and the development of magnetic nanoparticles, these structures are increasingly used in different applications. In the present study, we produced the recombinant human growth hormone from Pichia pastoris and for protein separation provided the surfaces similar to chromatographic columns on the surface of magnetic nanoparticles. For this purpose, using a co-precipitation method, the core of FeO was prepared and coated by silica. To increase the protein availability, silica mesoporous formation and its amine functionalization were performed. The specific surface area and the pore size were determined 78.3189 m/g and 7.44 nm. After the magnetic separation, the sample loading in SDS gel shows a reduction in protein band and the protein absorption at a wavelength of 280 nm. Finally, we evaluate the ability of amine functionalized nanoparticles for protein separation that demonstrate the adsorption capacity significantly increased compare with silica-coated nanoparticles. The amine functionalized nanoparticles provide the maximum adsorption capacity of 235.21 μg/mg and after the elution, protein concentration determined 476 mg/L. This work indicates the functionalized magnetic mesoporous silica nanoparticles can be used as the best candidate for the separation of different biological macromolecules.