This study describes a single gold nanoparticle (AuNP)-based observation of biomolecular interaction using a near-field scanning microscope (NSOM) in transmission mode. To observe streptavidin molecules, a glass surface was first patterned with a micro-scale line of (3-aminopropyl)trimethoxysilane (APTMS) by micro-contact printing (microCP) with a subsequent reaction of N-hydroxysuccinimide (NHS)-biotin. The AuNP-conjugated streptavidin was then applied to the biotin-modified glass surface and NSOM was employed to detect the resulting specific interaction between streptavidin and biotin on the glass surface. Using the optical and topological images generated from the NSOM analysis, the interaction could be observed at the nanoscopic scale. This study demonstrates that the NSOM is a powerful tool for the detection of protein interactions at the nanoscopic level when the protein is conjugated with AuNPs.