Biomolecular linear motor proteins -kinesin and microtubules -are finely patterned on a conventional, flat glass substrate using parylene lift-off process; these patterns are useful for single molecule analysis and biohybrid transportation. The patterning was performed from 5 μm 40 μm in width with minimal non-specific binding of the proteins. A conventional gliding assay was realized using this glass substrate and the behaviors of the single proteins were analyzed. © 2005 IEEE.