We report a new class of a SERS substrate with ordered nanostructures fabricated on silicon wafer using a deep UV (DUV) lithography technique followed by surface coating of silver and/or gold film. These substrates possess sharp edged nanogaps, which are responsible for the SERS enhancement. SERS performance of these substrates was analyzed by studying its reproducibility, repeatability and signal enhancement measured from 2-naphthalene thiol (NT) molecule covalently anchored on to the substrate. SERS performance of this substrate was also compared with a commercial substrate and metal film over nanosphere (MFON) substrate, which is one of the most promising reported substrates. It was found that MFON substrate showed a slightly higher SERS intensity among all three chosen substrates, but the relative standard deviation (RSD) of the intensity for the two prominent peaks of NT was about 7-14% while for our nanogap DUV substrate the RSD was less than 3% with comparable SERS signal intensities to MFON. For the commercial substrate, the relative standard deviation was about 7-9% but with a much lower SERS signal intensity. To our knowledge, this observed reproducibility along with good SERS enhancement with nanogap substrate is the best among the reported SERS substrates. These observed results with the nanogap substrate show great potential for the development of a sensitive SERS biosensing platform. Efficacy of the nanogap DUV substrate for biosensing was demonstrated for in vitro glucose sensing under physiologically relevant conditions. © 2010 Elsevier B.V.