Scanning electrochemical microscopy (SECM) was used for localized electrogeneration of hydroxyl radical to create patterns with diaphorase on the substrates immobilized with self-assembled monolayers (SAMs) of four different alkylsilane derivatives. Hydroxyl radical was generated at an SECM tip (microelectrode) in a solution containing H2O2 and Fe3+ by applying a reduction potential pulse of 0.00 V vs Ag/AgCl. The electrogenerated hydroxyl radical degraded the SAMs and removed them from the glass surfaces. Diaphorase patterns were formed on the substrates by physical adsorption onto the hydrophobic area or by chemical linkage to the hydroxyl radical-attacked area. Local diaphorase activity was visualized by SECM by detecting the diaphorase-catalyzed current of ferrocenylmethanol coupled with oxidation of reduced nicotinamide adenine dinucleotide.