This paper documents a study of an Au nano-dot array that was fabricated by electron beam lithography on a glass wafer. The patterns that had features of 100 nm dots in diameter with a 2-mum pitch comprised a total area of 200 x 200 microm(2). The dot-shaped Cr underlayer was open to the air after developing Poly(methyl methacrylate) (PMMA). When dipped into the Cr etchant, the exposed Cr layer was eliminated from the glass wafer in a short period of time. In order to ultimately fabricate the Ti/Au dot arrays, Ti and Au were deposited onto the arrays with a thickness of 2 and 40 nm, respectively. The lift-off procedure was carried out in the Cr etchant using sonication in order to completely remove the residual Cr/PMMA layer. The fabricated Au nano-dot array was then immersed in an Ag enhancing solution and then into an ethanol solution containing (N-(6-(Biotinamido)hexyl)-3'-(2'-pyridyldithio)-propionamide (Biotin-HPDP). The substrate was analyzed using a correlated atomic force microscopy (AFM) and confocal Raman spectroscopy. Through this procedure, position-dependent surface-enhanced Raman spectroscopy (SERS) signals could be obtained.