Here a novel technique is reported to construct a three-dimensional (3D) array of well-defined and controllable multilayered nanostructures of proteins that is based on alternate layer-by-layer assembly of bacterial protein nanoparticles and DNA on a patterned array of gold dots. This is the first report on protein-based multilayer stacking, which has the following significant advantages over conventional multilayer assemblies: 1) avoiding hazardous chemicals, the multilayer assembly is implemented in aqueous solution under mild temperature and pH conditions over a relatively short period; 2) direct multilayer growth from designated position is possible by controlling the aspect ratio; 3) multicomponent stacking can be easily performed through alternate stacking of different building blocks (in this case protein nanoparticles); and 4) a wide variety of 3D arrays can be constructed using various functionalized protein nanoparticles that are easily prepared through a simple genetic engineering approach. In this study, as a proof of concept, the developed 3D and patterned arrays of protein nanoparticle multilayers are successfully applied to the multiplexed bioassays of breast and colorectal cancer markers.