The adsorption of his-tag green fluorescent protein (GFPH6) onto the mica surfaces has been studied by atomic force microscopy (AFM) and laser confocal fluorescence microscopy. By controlling the adsorption conditions, separated single GFPH6 and GFPH6 monolayer can be adsorbed and formed on mica surfaces. In present experiments, based on the AFM measurement, we found that the adsorbed GFPH6 was bound on the mica surface with its β-sheets. The formed GFPH6 monolayer on mica surfaces was flat, uniform, and stable. Some applications of the formed monolayer have been demonstrated. The formed monolayer can be used as a substrate for DNA imaging and AFM mechanical lithography. Microsc. Res. Tech., 2008. © 2008 Wiley-Liss, Inc.