Three differently charged nanoscale features were constructed by sequential nanografting of 6-mercaptohexan-1-ol, N-(6-mercapto)hexylpyridinium bromide, and 3-mercaptopropionic acid into a self-assembled monolayer resist of an alkanethiol terminated with a hexa(ethylene glycol) group on an atomically flat template stripped gold surface. The immobilization of three proteins, lysozyme, rabbit IgG, and bovine carbonic anhydrase (II), onto these different charged nanopatches was studied at a variety of pH values. At pH 4.5, all three proteins adsorbed onto the charged nanosurfaces. At higher pH, the proteins behaved differently, depending on the pH and relative surface charge of the nanosurface. A surface charge distribution model was employed to explain the unusual adsorption behavior of carbonic anhydrase. Finally, an approach that combines electrostatic immobilization and specific protein-protein interactions to fabricate multiple-layered (protein G/rabbit IgG/anti-IgG) three-dimensional (3D) protein nanostructures is presented, demonstrating that the combination of nanografting, electrostatic immobilization, and specific protein interaction is a powerful tool for construction of novel 3D protein surface nanostructures.