Using the bianalyte method we unambiguously demonstrate that a single gold nanosphere on a metal film junction, in the so-called nanoparticle on a mirror configuration, is capable of single molecule detection with surface-enhanced Raman spectroscopy (SERS). Also this configuration serves as a convenient and highly sensitive SERS sensor for detection of biomolecules. Such simple nano-junction based systems are ideal for chemical and biomedical analysis.