This work reports a simple yet highly efficient method to fabricate transparent substrates for surface enhanced Raman scattering (SERS) application. Our technique is based on a fume etching process for commercial ITO-glass (ITO film on glass substrate) with atmospheric fume of hydrochloric acid (HCl). Surface analyses suggested that the spontaneously evaporated HCl molecules chemically reacted with ITO and formed island-like nanostructures. The topography of the ITO surface can be tuned to optimize the SERS efficiency by adjusting the etching parameters. On-line SERS spectra of the R6G solution concentration variation were also obtained to study the adsorption behavior of R6G on ITO surface. This convenient, simple, and low-cost method of making transparent SERS-active substrates potentially opens a way toward rapid, nondestructive and quantitative bioanalysis, particularly in microfluidics. (C) 2014 Elsevier B.V. All rights reserved.