Characterization of cytochrome c (Cyt C) bound to self-assembled monolayers (SAMs) of mercaptoalkanoic acids (CnCOOH) on colloidal silver was carried out by means of surface-enhanced resonance Raman spectroscopy. Cyt C selectively adsorbed on negatively charged surfaces of SAMs on Ag, and a clear surface-enhanced resonance Raman scattering (SERRS) from Cyt C superimposed on surface-enhanced Raman scattering (SERS) from SAMs was observed under 514.5 nm excitation (in resonance with the electronic absorption of Cyt C (beta band)). Whereas, in the spectrum of the same sample under 488.0 nm excitation (off resonance with the electronic absorption of Cyt C), any signals attributable to Cyt C were not observed, but only those attributable to CnCOOH were detectable. These observations strongly suggest that Cyt C makes a second layer on a SAM which is directly attached to Ag. Comparing the surface-enhanced resonance Raman spectra with the resonance Raman spectra in solution revealed that the native structure of Cyt C was fully preserved after adsorption on a SAM, while adsorption-induced structural change took place in Cyt C which was directly adsorbed on Ag. It was concluded that Ag modified with a SAM is a suitable SERS-active substrate to analyze the structure of proteins on biomimetic membrane surfaces without denaturation.