We propose and experimentally demonstrate a method of active immobilization for biomolecules on a three-dimensional nanometre-scale electrode (3D nanoelectrode) using dielectrophoresis to immobilize the biomolecules at predetermined locations for single-biomolecule study. We have developed a novel two-step fabrication process for obtaining a 3D nanoelectrode having a sharp top, which is necessary for immobilizing a single biomolecule at a single point. The first step is to fabricate the backbone structure, which is rigid and defines the shape of the 3D nanoelectrode. It was fabricated with diamond-like carbon (DLC) obtained using focused ion beam assisted chemical vapour deposition followed by post-plasma etching, which reshapes the DLC structure. The second step coats the DLC structure with a thin layer of aluminium, which supplies electrical conductivity to the DLC structure. By applying a high frequency (of the order of megahertz) and high intensity (greater than or equal to a few megavolts per metre) electric field using the 3D nanoelectrodes, the generated dielectrophoresis attracted and then immobilized target biomolecules onto the tops of 3D nanoelectrodes, as a demonstration of active immobilization of biomolecules.