Bio-functionalized gold nanoparticles (AuNPs), which bio-specifically interact with biotin-(strept)avidin, were investigated in this study. AuNPs were functionalized with a synthetically-provided biotin-linked thiol (BLT), which was synthesized by amidation of the active ester of biotin with 2-mercaptoethylamine. The BLT-attached AuNP was bio-specific for streptavidin, making it potentially useful for biosensor applications. To test the bio-specific interactions, the colors, absorption spectra and TEM images were investigated for proteins such as streptavidin, cytochrome C, myoglobin and hemoglobin. The colors and absorption spectra changed when streptavidin was added to the BLT-attached AuNP solution. However, the color and spectra did not change when the other proteins were added to the same solution. These results show that the AuNPs provided a colloidal solution with excellent stability and highly selective absorption characteristics for streptavidin as a target molecule. Proteins were also screened in order to identify a general strategy for the use of optical biosensing proteins based on AuNPs. In addition, TEM images confirmed that streptavidin led the BLT-attached AuNPs to aggregate or precipitate.