Surface-enhanced Raman spectroscopy (SERS) has received renewed interest in recent years in fields such as trace analysis, biorelated diagnosis, and living cell study. However, the interference of impurities left on the surface from the preparation process of substrates or adsorbed from the ambient environment limits to some extent the application of SERS for analysis of trace or unknown samples. In the present paper, we propose a method to prepare clean SERS substrates by a combined method of chemical adsorption of iodide on the Au surface to remove the surface impurities and electrochemical oxidation of the adsorbed iodide to obtain a clean and impurity-free surface for SERS measurement. Time-dependent control experiment of untreated and treated substrate reveals that this method is very effective in obtaining substrates free of impurities. SERS mapping demonstrates the SERS activity of the substrate is homogeneous over the whole surface. The obtained clean substrate enables us to study the structures of the cell membrane with SERS and even to perform SERS mapping to visualize the distribution of amino acids over the membrane of a living cell.