In this study, surface-enhanced Raman scattering (SERS) in combination with a heat-induced sensing technique has been applied for investigating molecular orientations of glutathione molecules adsorbed on silver colloidal nanoparticles under different chemical environments, which has enabled us to further study how glutathione molecules are adsorbed on the silver surfaces. Factors that may affect the configurations of glutathione molecules adsorbed on the silver nanocolloids have been investigated. By observing the relative enhancement factors of SERS bands due to individual functional groups contributed from different terminals, the affinity between the different functional groups of glutathione and the silver surfaces under different conditions has been sorted and the orientations of glutathione molecules adsorbed on the silver surfaces have been investigated.