The magnetite (Fe3O4) nanoparticles with immobilized metal affinity ligands were prepared for protein adsorption. First, magnetite nanoparticles were synthesized by hydrothermal method. Charged with Zn2+, the magnetic nanospheres were applied to adsorb bovine hemoglobin (BHb). The morphology and magnetic properties of magnetic nanospheres were investigated by TEM. These magnetic nanospheres modified with metal ligands can be used efficiently in protein affinity separation and purification.