The disassembly of a core-satellite nanostructured substrate is presented as a colorimetric biosensor observable under dark-field illumination. The fabrication method described herein utilizes thiol-mediated adsorption and streptavidin-biotin binding to self-assemble core-satellite nanostructures with a sacrificial linking peptide. Biosensing functionality is demonstrated with the protease trypsin, and the optical properties of the nanoassemblies are characterized. A figure of merit is presented to determine the optimal core and satellite size for visual detection. Nanoassemblies with 50 nm cores and 30 or 50 nm satellites are superior as these structures achieve an orange to green color shift greater than 70 nm that is easily discernible by the naked eye. This colorimetric substrate may prove to be a favorable alternative to liquid-based colloidal sensors and a useful visual readout mechanism for point-of-care microfluidic diagnostic assays.