We used the Langmuir-Blodgett (LB) method for preparation of large area, defect-free monolayer silica particle film as a template for the fabrication of nano-structure Au pattern on a conventional Au substrate for Surface Plasmon Resonance (SPR). Well organized, trigonal pyramid shaped Au nano-structures were able to construct on 34 separate chips in one fabrication process. The dimensions of trigonal pyramid nano-structures were precisely controlled by changing the particle size of the silica LB template. The nano-structure Au patterned SPR chips provides the enhancement of sensitivity in SPR analysis. The sensitivity enhancement of nano-structure patterned chips was evaluated by comparison with conventional flat Au chips. The nano-structure patterned substrate demonstrated a sensitivity enhancement up to 120% compared to a conventional SPR chip when ethanol solution was used as an analyte.