 We describe here a sensitive sandwich-type electrochemiluminescence DNA biosensor based on a polyaniline–nanogold (PANI–AuNPs) modified platform with tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate ([Ru(bpy)3]2+)–nanoporous gold (Ru–NPG) composite as a label. The PANI was obtained by electropolymerization of aniline on the surface of indium–tin oxide (ITO) glass and gold nanoparticles (AuNPs) were fabricated by the reaction of PANI. The fabricated AuNPs were further grown to a larger size in HAuCl4 solution and the capture DNA was fastened onto the ITO glass on the basis of the interaction with AuNPs. NPG was prepared by a selective dissolution of silver from silver–gold alloy in nitric acid, which provided free-standing noble metal membranes with controllable three-dimensional porosity to immobilize more [Ru(bpy)3]2+. On the basis of a sandwich-type hybridization reaction, the Ru–PANI labeled reporter DNA was captured by the target DNA. The as-proposed DNA biosensor enables target DNA concentrations to be determined in the 0.05–1,000 fM concentration range, with a detection limit of 0.01 fM.