Objective：To prepare and characterize magnetic microspheres coated with copolymers of two unsaturated esters of Polyethylene glycol（PEG）. Methods：Fe3O4 nanoparticles were prepared by a chemical co-precipitation method.Magnetic microspheres were prepared by coating Fe3O4 cores with copolymers of monomethoxyl-PEG 350 acrylate and the single maleic acid ester of PEG 1540. Hydroxyl groups on the surfaces were converted to methanesulfonate that was reacted with ethylene diamine；the resulting amino groups were modified with an activated biotin ester. Microsphere sizes were examined by transmission electronic microscopy. The specific bindings were tested with streptavidin labeled by alkaline phosphatase；the nonspecific bindings were tested with 4-nitro-1-naphthylol benzoate and analyzed via HPLC-UV. Results：Fe3O4 cores had the diameters of about 60 nm with wide distributions while the biotinylated magnetic microspheres had the diameters of about 200 nm. The biotinylated magnetic microspheres selectively recov－ered the labeled streptavidin from the lysate of E.coli with a binding capacity of about 51 ng protein/g magnetic microspheres.4-nitro-1-naphthylol benzoate at low concentrations had negligible bindings; its binding ratio increased but still had no significance even at 64 μmol/L. Conclusion：Hydrophilic magnetic microspheres prepared by coating Fe3O4 cores with copolymers of two unsaturated es－ters of PEG can be functionalized to exert the designed specific interactions while negligible nonspecific interactions with hydrophobic compounds，but the binding capacity needs further enhancement.