Surface Enhanced Raman Scattering (SERS) technique is used as an indispensable and sensitive modality for bio-sensing due to its ability to distinguish the analyte molecules based on their distinct 'fingerprint' spectra. One of the most promising SERS substrates for biosensing was fabricated by coating noble metal film over orderly packed nanospheres. However, the major challenge in developing such a sensor is to achieve reproducible SERS substrate. Here, we report a new class of SERS substrate with ordered 3D nanostructures fabricated on silicon wafer by deep UV lithography technique followed by bi-metallic coating of silver and gold. Compared to the substrate fabricated by conventional nanosphere lithography, this approach allows better control of the nanostructures, which in turn gives uniform surface roughness for the metal film to provide adequate SERS enhancement with high reproducibility. Significance of this substrate for biomedical application was demonstrated by glucose sensing under physiologically relevant conditions. Partitioning and localization of glucose molecules within the first few nanometers of active SERS substrate was achieved by a self assembled monolayer (SAM) on the surface of substrate. © 2009 Copyright SPIE - The International Society for Optical Engineering.