In this study, non-resonant second harmonic generation (SHG) was used to investigate the molecular structure and adsorption of DMSO at the air/neat DMSO liquid and air/DMSO aqueous solution interfaces. The molecular orientation of interfacial DMSO as a function of the bulk DMSO concentration was investigated by quantitative polarization SHG analysis. For the air/neat DMSO liquid interface, the transition dipole moment of the S[double bond, length as m-dash]O group of DMSO is oriented 140° from the surface normal, where the S[double bond, length as m-dash]O group of DMSO is estimated to be 30° from the surface plane. The orientation of the S[double bond, length as m-dash]O group of interfacial DMSO is not dependent on the bulk DMSO concentration. Furthermore, the concentration-dependent SHG signal confirmed that the antiparallel double layer structure does not form at the air/DMSO water interface. The free energy of adsorption of DMSO at the air/DMSO aqueous solution interface was determined to be ΔGads = -5.6 ± 0.4 kJ mol-1.