A versatile approach based on nanosphere lithography is proposed to generate surface-imprinted polymers for selective protein recognition. A layer of 750 nm diameter latex bead-protein conjugate is deposited onto the surface of gold-coated quartz crystals followed by the electrosynthesis of a poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT/PSS) film with thicknesses on the order of the bead radius. The removal of the polymer bead-protein conjugates, facilitated by using a cleavable protein-nanosphere linkage is shown to result in 2D arrays of periodic complementary size cavities. Here it is demonstrated by nanogravimetric measurements that the imprinting proceeds further at molecular level and the protein (avidin) coating of the beads generates selective recognition sites for avidin on the surface of the PEDOT/PSS film. The binding capacity of such surface-imprinted polymer films is ca. 6.5 times higher than that of films imprinted with unmodified beads. They also exhibit excellent selectivity against analogues of avidin, i.e., extravidin, streptavidin, and neutravidin, the latter being in fact undetectable. This methodology, if coupled with properly oriented conjugation of the macromolecular template to the nanoparticles, offers the possibility of site-directed imprinting.