A novel immobilization method of cupredoxin azurin on a gold surface was developed without a chemical linker using recombinant technique. A recombinant protein with cysteine residue by site-directed mutagenesis (SDM) was designed and then directly self-assembled on Au surface. The layer of the functionalized protein immobilized is confirmed by surface plasmon resonance (SPR) and its surface morphology is analyzed by scanning tunneling microscopy (STM). The immobilization efficiency has been increased about 73.2%, as compared to that of wild type azurin. The electrochemical property of the fabricated thin layer was investigated by the cyclic voltammetry (CV). As a result, cysteine-substituted azurin can be used for making high quality protein film, and applied to the fabrication of nano-scale bioelectronics.