For developing a magnetic bioassay system, an investigation to determine the presence of a specific biomolecular interaction between biotin and streptavidin was done using magnetic nanoparticles and a silicon substrate with a self-assembled monolayer. Streptavidin was immobilized on the magnetic particles, and biotin was attached to the monolayer-modified substrate. The reaction of streptavidin-modified magnetic particles on the biotin-modified substrate was clearly observed under an optical microscope. The magnetic signals from the particles were detected using a magnetic force microscope. The results of this study demonstrate that the combination of a monolayer-modified substrate with biomolecule-modified magnetic particles is useful for detecting biomolecular interactions in medical and diagnostic analyses.