In this paper, the surface-enhanced Raman scattering (SERS) active substrates of indium tin oxide (ITO) were prepared on aluminum foil by laser pulse deposition and vacuum annealing, and the SERS characteristics of ITO substrates were investigated. Five substrates with pulse numbers of 700, 1 000, 1 300, 1 600 and 2 000 were deposited. The measurements show that the film thickness is close to the linear relationship with the pulse number. When the thickness of ITO film is 60.80 nm with the pulse number of 1 300, the enhancement degree of Raman signal reaches the maximum, and the Raman intensity of the prepared ITO substrate is about 2 to 3 times that of the Au substrate. The results show that vacuum annealing can significantly improve the Raman enhancement effect of ITO substrates. All substrates with different thicknesses of ITO films have obvious SERS enhanced effect. SERS enhancement effect of the ITO substrates can be adjusted by varying the film thickness. These results provide a reference for subsequent SERS research and application of indium tin oxide.