Surface-enhanced Raman scattering (SERS) is an ultra-sensitive spectroscopic technique which has great application prosepct in characterization of dynamic molecular structures. Self-assembled monolayers (SAMs) of alkanethiolates are a typical kind of ultrathin films which have tremendous applications in the fields of bionics, materials, electronics, biology and chemistry. In the present study, the SAM structures of 1-hexanethiol when adsorbing on Ag nanoparticles were explored. The vibrational modes of trans (T) and gauche (G) conformations were employed to illustrate the molecular structures. The intensity ratios of T and G bands concerning to C—S, C—C and CH3 groups indicate the crystallinity of the SAMs. The results show that when HT is in high concentrations the crystalline process is quick, while in low concentrations, the crystalline process is very slow. The dynamic study on the formation of SAM films is of great significance for the corrosion protection, device fabrication, and sensor applications.