A simple method for constructing gold nanoparticle-modified electrodes with three-dimensional nanostructures is demonstrated. The electrodes were prepared by casting citrate-reduced AuNPs onto polycrystalline gold electrodes. The resultant electrodes had a large surface area-to-volume ratio, adequate for high protein loading and conferring high stability. The gold nanoparticle electrodes were covered with a self-assembled monolayer of 11-mercaptoundecanoic acid for electrostatic immobilization of cytochrome c (cyt c). At the electrode, direct, reversible electron transfer from cyt c was observed with remarkable stability. Moreover, an extremely high surface coverage of electrochemically active cyt c, 167 fully packed monolayers, was obtained through use of the electrode.