The fabrication method of nanoscale myoglobin monolayer using chemical linker is introduced in this study because control of amount and orientation of protein immobilized on electronic device is one of main issues to be solved for the realization of biomolecular electronic device. Myoglobin, metalloprotein, is selected as active material due to its electrochemical property. To immobilize myoglobin on Au surface, 3,3-dithiobis (sulphosuccinimidyl propionate) (DTSSP) is utilized as a chemical linker. The optimum amount of protein is investigated by surface plasmon resonance (SPR). SPR and scanning tunneling microscope (STM) results confirm the nano scale protein layer formed on DTSSP self assembled monolayer (SAM) on Au surface. Protein layer on Au surface using DTSSP as chemical linker was more stable than random adsorption without linker as aspect of redox character due to the fact that myoglobin immobilized with chemical linker did not lose its redox property after long usages.