New approach to create a pattern of specific protein binding sites via nanoimprint lithography is presented. NH3-plasma functionalized, patterned polystyrene covalently binds a biotin linker to which streptavidin is bound. The streptavidin is used as a versatile anchor for biotinylated functional proteins by means of a sandwich-type quantitative fluorescence immunoassay. Rabbit IgG is the analyte used as a "proof of concept" for the designed system, yielding a detection limit of approximately 50 ng/ml.