Surface-enhanced Raman scattering (SERS) technology, which enables the rapid detection of trace molecules, has gained widespread attention in areas such as pharmaceutical material analysis, identification of hazardous substance residues, and food safety. The response limit of SERS to the detected substances depends on the performance of the substrate and directly affects the final detection effect. In this paper, MoO3-x nanoslots with oxygen vacancy defects were successfully prepared by a simple hydrothermal method with the aim of increasing the specific surface area of the substrate and its ability of photoinduced charge transfer between the substrate and the detected substances. The electronic structure, optical properties and surface chemical environment of the MoO3-x nanoslots were analyzed by a combination of X-ray photoelectron spectroscopy (XPS), ultraviolet-visible spectroscopy (UV-Vis) and Fourier transform infrared spectroscopy (FT-IR). Three organic dye molecules, rhodamine 6G (R6G), crystalline violet (CV) and rose red B (RhB), were detected using the MoO3-x nanoslots as SERS substrates, and the experimental results show that the detection limit of R6G could reach 10-10 mol/L, and it has better Raman-enhancing activity compared with the same type of semiconductor substrates.