Thymosin‐β4 is a peptide found at high concentrations in a wide range of mammalian cells. It has been shown to be elevated in metastatic cells. In this study, the surface‐enhanced Raman spectrum of thymosin‐β4 was obtained using a silver nanorod array surface‐enhanced Raman scattering substrate. Significant Raman peaks were found, and the corresponding vibrational modes were assigned based on previously published literature, with amide A-B, amide I, and amide III modes all identified. The majority of the remaining peaks were assigned modes based on the spectra of lysine and glutamic acid, the two most abundant amino acids in the thymosin‐β4 sequence. A standard Raman spectrum of thymosin‐β4 was also obtained and analyzed for comparison. Because thymosin‐β4 plays a significant role in regulating the formation of cellular cytoskeleton and other several biological processes, its Raman spectrum will be of use to researchers.