Molecularly imprinted polymer (MIP) films on silicon wafers were prepared by surface-initiated atom transfer polymerization (ATRP). 2-hydroxyethyl methacrylate and ethylene glycol dimethacrylate were used as the functional monomer and the cross-linking agent, respectively. Myoglobin (Mb) was selected as the template molecule to form the MIP films. Various characterization techniques including ellipsometry, contact-angle goniometer, atomic force microscopy, grazing angle-Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, were used to characterize the films. Ellipsometry was also used to quantify and compare the binding capacity of the MIP and nonimprinted polymer (NIP) films. The MIP films exhibited higher rebinding capacity than the NIP films at all solution concentrations of Mb. A selectivity coefficient of 3.15 was achieved for the MIP films prepared against the Mb template molecule.