To spread cytochrome c, the water soluble protein, on subphase homogeneously, cytochrome c buffer solution was mixed with ethanol diluted with a deionized distilled water (DDW). After spreading it, cytochrome c multi-layers were deposited onto ITO coated glass by Schaefer technique (the horizontal lifting technique), and Langmuir-Blodgett (LB) technique (vertical lifting technique). Cytochrome c multi-layers deposited onto the substrate by Schaefer technique and LB technique were analyzed by UV-visible spectroscopy, and multi-layers of cytochrome c fabricated using the Schaefer technique was analyzed by Atomic Force Microscopy (AFM).