Holographic lithography has been an efficient and versatile method to fabricate nanosize patterned devices during past several years. This technique is based on the interference of UV-laser beams for patterning regular arrays of fine features without the use of mask as used in conventional lithography. Simple gratings and two-dimensional dot arrays has been fabricated by using the interference principle. Using this technique, we fabricated line-shaped embossing gold (Au) structure on silicon substrates. Towards the characterization of fabricated line-shaped Au nano-structure Field Effect Scanning Electron Microscopy (FE-SEM) is used and our results clearly show the fine structure of fabricated Au lines. Further characterization towards our patterned substrates we used Atomic Force Microscopy (AFM) for analysis of Au patterned lines. A bioelectronic device consisting of cysteine modified azurin was fabricated where the protein was covalently immobilized on the nano-patterned substrate and we investigated the electrochemical property of the device by Cyclic Voltammetry (CV) experiments comparing with that of a normal flat Au substrate. We think these nano-patterned Au substrates can be applied as potentially usable elements for the development of biosensor and other bioelectronic devices.