The fundamental characteristic of nanotechnology is the fabrication of structures with molecular dimensions. In connection with a novel immunosensor, a specifically tailored molecular environment is required which combines immobilized biochemical recognition with electrochemistry. These demands are fulfilled by the creation of nanometer-size electrodes in a thin film (similar to 30 nm) of gold consisting of openings commensurate with the size of the biomolecule (similar to 10 nm). We report on three complementary and advanced fabrication techniques used to produce closely packed distributions of these features : direct focused ion beam milling, lift-off using polystyrene latex nanospheres as a masking material, and resist exposure by individual ionization tracks produced during high energy heavy ion (similar to 0.1 MeV/amu) irradiation. The latter two processes are stochastic in nature and exploit the fact that no precise arrangement of the openings is necessary for this application. Fabrication results were analyzed by scanning electron and scanning force microscopy. The three methods are compared with respect to opening size, definition, and packing density and process throughput and control. While nanosphere lift-off represents a relatively simple and reproducible structuring technique, heavy ion irradiation has the potential for achieving the smallest openings (similar to 15 nm). Ultimately, the choice of nanostructuring method is dictated by the electrode’s performance in the sensor.