A key aspect of biochip and biosensor preparation is optimizing surface attachment of biomolecules. Here, we report a facile approach for selectively immobilizing biomolecules on amphiphilic polymer-coated plastic surfaces with anti-biofouling properties. To modify plastic surfaces, we synthesized two types of random copolymers by radical polymerization, which consisted of three parts: an anchoring group; a PEG component, which acted as a repellent of nonspecific biomolecules; and a functional group, to which biomolecules were conjugated. Dodecyl- and benzyl-based copolymers were highly soluble in water, presumably due to the presence of multiple PEG groups, and could easily coat the model plastic surface (polystyrene) in an aqueous environment. The antibiofouling property of each polymer-coated plastic surface was examined by measuring the extent of nonspecific protein adsorption using bovine serum albumin (BSA). Both polymer-coated plastic surfaces showed a very low level of BSA adsorption relative to that of an uncoated plastic surface (control). Finally, we showed that streptavidin and antibodies, as representative biomolecules, could be selectively immobilized on the polymer-coated plastic surfaces imprinted with biotin and protein A, respectively, by microcontact printing, exhibiting an intense signal with low background.