The applicability of surface-enhanced Raman spectroscopy is demonstrated to probe the adsorption behavior of individual molecules on a Ag electrode. High-quality SERS spectra of (R)-di-2-naphthylprolinol (DNP) were obtained from ultradilute solutions (10−12 M) on the Ag-nanoparticle-modified Ag electrode, which is attributed to the high electromagnetic (EM) effect of the SERS-active system as well as to the strong adsorption and interaction of DNP molecules with Ag. The stable SERS spectra present remarkable potential dependence, which gives evidence for the behavior of individual DNP molecules on the Ag surface. Based on statistical analysis for the probability of DNP molecules located in ‘hot spots’, we propose an SERS mechanism for individual molecules in the electrode system, in combination with the hot-spot model and orientation of the probe molecules. Copyright © 2010 John Wiley & Sons, Ltd.