The capability of assembling biomotors onto specific locations of solid substrates is a key for development of biomotor-based nanomechanical systems. We developed a method to direct the assembly of the heavy meromyosin fragment from rabbit skeletal muscle myosin onto specific locations of Au substrates utilizing surface molecular patterns. In this strategy, chemically directed patterns of streptavidin were achieved to direct highly specific assembly of biotinylated heavy meromyosin on the substrates-a strategy applicable for patterning a variety of biotinylated molecules-while BSA was utilized to avoid nonspecific adsorption. In vitro motility assays of filament sliding were used to confirm functionality of assembled actomyosin.