A novel approach for micropatterning of surfaces with organic and biological microstructures using the scanning electrochemical microscope (SECM) is described. The approach is based on the introduction of the spatial resolution by local deposition of gold particles followed by monolayer formation and functionalization. Specifically, gold patterns were deposited locally on silicon wafers with the SECM as a result of the controlled anodic dissolution of a gold microelectrode. The gold patterns were further used as microsubstrates for assembling cystamine monolayers to which either fluoresceine isothiocyanate (FIT) or glucose oxidase (GOD) were covalently attached. Characterization of the organic monolayers, as well as the biological activity of the enzyme patterns, was carried out by fluorescence microscopy and the SECM, respectively.