In this study, a novel route for the preparation of magnetite (Fe(3)O(4)) nanoparticles (NPs) with immobilized metal affinity ligand iminodiacetic acid (IDA) charged with Cu(2+) was developed. First, magnetite nanoparticles were synthesized by a hydrothermal method. Charged with Cu(2+), the magnetic nanoparticles (MNPs) were applied to separate a model protein mixture of bovine hemoglobin (BHb) and bovine serum albumin (BSA). They could be separated completely and showed low non-specific adsorption. The morphology, structure and composition of the magnetite MNPs were characterized by transmission electron microscopy, power x-ray diffraction, x-ray photoelectron spectrometry and Fourier transform infrared spectroscopy. The resulting magnetite MNPs charged with Cu(2+) show not only a strong magnetic response to externally applied magnetic field, but are also highly specific to protein BHb. It is interesting that MNPs modified with metal ligands showed a property of magnetic colloid photonic crystals. Furthermore, they could efficiently remove the abundant protein bovine hemoglobin from bovine blood. They have potential application in removing abundant protein in proteomic analysis.