In this study, the Raman spectral characteristics of 1,4-phenylenediisocyanide (1,4-PDI) and 4-aminobenzenethiol (4-ABT) positioned at the nanogap formed by Au/Ag alloy nanoparticles and a flat Au substrate were examined, and three-dimensional finite-difference time-domain (3-D FDTD) calculations were carried out. A more intense Raman signal was measured, regardless of the excitation wavelength, when Ag-rich Au/Ag alloy nanoparticles were used to form the nanogaps. Regarding the excitation wavelength, 568 nm of light was the most effective in inducing a Raman signal, particularly when Ag nanoparticles were adsorbed on 1,4-PDI or 4-ABT, whereas 632.8 nm of light was slightly more effective than 568 nm of light when Au nanoparticles were adsorbed onto them. The Raman spectra of 1,4-PDI could be attributed to the electromagnetic enhancement mechanism. The dependencies of the Raman spectra of 1,4-PDI on the excitation wavelength and the type of Au/Ag alloy nanoparticle were comparable to those predicted by the 3-D FDTD calculations. From the measured NC stretching frequencies, the surface of 35 nm sized Au/Ag alloy nanoparticles containing more than 5 mol % of Ag atoms was concluded to be covered fully with Ag atoms. The Raman spectra of 4-ABT were interpreted to be a product of electromagnetic and chemical enhancement mechanisms. Assuming that the Raman intensity ratios of the b 2 -and a 1 -type bands were indicative of the extent of chemical enhancement, the Ag-to-4-ABT electron transfer appeared more facile than the Au-to-4-ABT transfer did and more favorable by excitation with a 514.5 nm laser than a 568 nm or 632.8 nm laser.