In this study, surface-enhanced Raman scattering (SERS) spectra of 6-propylthiouracil (6-PTU) on Au surface and the interaction between 6-PTU and human serum albumin (HSA) were studied. The Raman bands were assigned by the density functional theory calculations at the B3LYP/6-311++g(d,p) level. Furthermore, the effects of concentration on the SERS spectra of 6-PTU were analyzed. It shows that with the changes of the concentrations of 6-PTU, the SERS spectra of 6-PTU present significant changes, and it can be concluded that with the changing of concentrations, the orientation of 6-PTU on Au surface also changes. In addition, the SERS spectra of the interaction between 6-PTU and HSA show that the binding sites of 6-PTU to HSA are the functional groups N3H and C¼O. The information will not only be references to the study of the mechanism of the interaction between drugs and blood plasma or serum albumin but also a guidance to understand the metabolism of drugs in human body.