A novel and uncomplicated synthesis method of Cu2+-chelating with carboxyl groups that directly-modified NiFe2O4 magnetic microspheres (NiFe2O4-PAA-Cu2+) was fabricated for selective enrichment and separation of bovine hemoglobin (BHb). First, a carboxyl group directly-modified on NiFe2O4 magnetic microspheres was gained through a facile one-pot solvothermal method. Second, Cu2+ from CuSO4 was brought into use to react with carboxyl groups under mechanical stirring at room temperature. The resulting magnetic microspheres were characterized by distinct instruments that included transmission electron microscopy (TEM), Brunauer-Emmett-Teller (BET), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), vibrating sample magnetometer (VSM) and scanning electron microscope (SEM) to examine the size, morphology, composition and magnetization characterization. The results indicated that the NiFe2O4-PAA-Cu2+ microspheres exhibited good saturation magnetization(36.686 emu g?1), which can facilitate magnetic separation under the help of an outside magnetic field. Also, good dispersion and high adsorption ability to BHb (783.53mg g?1) can be applied to selective enrichment for bovine hemoglobin and used for selective sorption of BHb protein in bovine blood samples.