A novel and effective protocol for the surface modification and quantitative characterization of magnetic polymeric nanospheres prepared by miniemulsion polymerization is reported. Composite nanospheres consisting of polymer-coated iron oxide nanoparticles were prepared by the miniemulsion polymerization of methyl methacrylate and divinylbenzene in the presence of magnetic fluid. Surface modification reaction of the magnetic polymer with poly(ethylene glycol) (PEG) was employed to obtain a hydrophilic hydroxyl-group-functionalized magnetic nanospheres. An affinity dye, Cibacron blue F3G-A (CB), was then coupled covalently to prepare a magnetic nonporous affinity adsorbent. The morphology and magnetic property of the polymer nanospheres obtained were examined by transmission electron microscopy and a vibrating sample magnetometer. The contents of surface groups modified were quantitatively measured by using diffusive reflectance Fourier transform infrared spectroscopy on the basis of a linear relationship between the intensity ratio of IC-O-C/I-C=O and the content of PEG. X-ray photoelectron spectroscopy (XPS) was used to examine the surface of magnetic nanospheres. It was confirmed by the comparison of XPS spectra of both dye-coated and uncoated magnetic nanospheres to which the CB ligand was coupled, and the surface of the PEG-modified nanospheres had an exact 3:7 atomic ratio of sulfur to nitrogen.