This paper describes a technique developed for the formation of the self-assembled protein monomolecular layer. The main idea is a direct transfer of protein molecules consisting of a Langmuir-Blodgett (LB) film onto the surface of another chemically modified metal substrate and induction of the spontaneous formation of a self-assembled monolayer (SAM) by chemisorption without protein aggregates. In the present experiments, a cytochrome c (cyt c) SAM on a gold substrate was prepared by incubating a EDC/MUA-modified gold substrate overlaid with a cyt c LB film in a phosphate buffer solution. Scanning tunneling microscopy (STM) image of a cyt c SAM shows that the size of cyt c clusters in the most part of the substrate is approximately 5 nm, indicating the cyt c monomolecular layer. The rectifying property of a cyt c monolayer was confirmed to remain by an asymmetric I-V curve in the applied bias from -1 V to 1 V.