The self-assembled monolayers (SAMs) of 6-mercaptopurine (6MP) were formed at the roughened polycrystalline gold surfaces in acid and alkaline media. The time-dependent Raman mapping spectral analysis in conjunction with the quantum calculations for the vibrational modes using ab initio BLYP/6-31G method suggested that both of the resulted 6MP SAMs adopted the same adsorption mode through the S atom of pyrimidine moiety and the N7 atom of the imidazole moiety anchoring the gold surface in a vertical way. The in situ surface-enhanced Raman scattering spectroelectrochemical experiment was conducted to examine the stability of the SAMs at various bias potentials. It was found that the detaching process of the 6MP SAMs from the surface involved one electron reduction as the voltage was applied at ca. 0.7 V vs a standard calomel electrode.