Diaphorase was hydrophobically immobilized onto a self-assembled monolayer of alkanethiol at an interdigitated Au microarray. The diaphorase activity at the array substrates was imaged with the scanning electrochemical microscopy (SECM) in the presence of NADH and ferrocenemethanol as a redox mediator. The images demonstrate that diaphorase was immobilized onto the SAM layer to form a line-and-space micropattern which reflected the pattern of the Au microarray.