The present study reports the covalent immobilization of myosin on glass surface andin-vitro motility of actin-myosin biomolecular motor. Myosin was immobilized on poly-L-lysine coated glass using heterobifunctional cross linker EDC and characterized by AFM. Thein-vitro motility of actin was carried out on the immobilized myosin. It was observed that velocity of actin over myosin increases with increasing actin concentration (0.4–1.0 mg/ml) and was found in the range of 0.40–3.25 μm/s. The motility of actinmyosin motor on artificial surfaces is of immense importance for developing nanodevices for healthcare and engineering applications