A process to structure gold electrodes with nanometer-sized dimensions for biosensor applications has been developed. Latex spheres (60 nm diam) are used as a masking material during the evaporation of a gold film onto a Si/SiO2 substrate. Openings left in the film after lift-off of the spheres are suitable in size to immobilize proteins such as antibodies or enzymes which can art as specific recognition elements. The nanometer-scale proximity of the recognition elements to the conducting material allows the development of mediatorless biosensors. This paper describes the optimization of the nanostructuring process as well as the morphological and electrochemical characterization of the structured electrodes.