Functional polymer brush nanostructures are obtained by combining step-and-flash imprint lithography (SFIL) with controlled, surface-initiated polymerization (CSIP). Patterning is achieved at length scales such that the smallest elements have dimensions in the sub-100 nm range. The patterns exhibit different shapes, including lines and pillars, over large surface areas. The platforms obtained are used to selectively immobilize functional biomacromolecules. Acrylate-based polymer resist films patterned by SFIL are first used for the selective immobilization of ATRP silane-based initiators, which are coupled to unprotected domains of silicon substrates. These selectively deposited initiators are then utilized in the controlled radical SIP of poly(ethylene glycol) methacrylates (PEGMA). Nanostructured brush surfaces are then obtained by removal of the resist material. The areas previously protected by the SFIL resist are passivated by inert, PEG-based silane monolayers following resist removal. PEGMA brush nanostructures are finally functionalized with biotin units in order to provide selective attachment points for streptavidin proteins. Atomic force microscopy and fluorescence spectroscopy confirm the successful immobilization of streptavidin molecules on the polymer grafts. Finally, it is demontrated that this fabrication method allows the immobilization of a few tens of protein chains attached selectively to brush nanostructures, which are surrounded by nonfouling PEG-functionalized areas.