It is known that protein attachment to surfaces depends sensitively upon the local structure and environment of the binding sites at the nanometer scale. Using nanografting and reversal nanografting, both atomic force microscopy (AFM)-based lithography techniques, protein binding sites with well-defined local environments are designed and engineered with nanometer precision. Three proteins, goat antibiotin immunoglobulin G (IgG), lysozyme, and rabbit immunoglobulin G, are immobilized onto these engineered surfaces. Strong dependence on the dimension and spatial distribution of protein binding sites are revealed in antibody recognition, covalent attachment via primary amine residues and surface-bound aldehyde groups. This investigation indicates that AFM-based nanolithography enables the production of protein nanostructures, and more importantly, protein-surface interactions at a molecular level can be regulated by changing the binding domains and their local environment at nanometer scale.