The development of cost-effective methodologies for the precise nanometer-scale positioning of biomolecules permits the low-cost production of various biofunctional devices for a range of biomedical and nanotechnological applications. By combining colloidal lithography and the mussel-inspired multifunctional polydopamine coating, we present a novel parallel benchtop method that allows rapid nanoscale patterning of proteins without the need for electrically powered equipment in the fabrication process. The PDA-immobilized binary nanopattern consisting of BSA surrounded by PLL-g-PEG is fabricated over a large area, and the integrity of the pattern is confirmed using AFM and FM.