In recent years, surface-enhanced Raman scattering (SERS) based on metal oxide semiconductors has been an active area of research and development, attracting significant scientific interest. These SERS substrates are known as plasmon-free SERS substrates because they are not based on noble metal nanoparticles but mainly on the defects, structure, and surface morphology of semiconductors to enhance the Raman signal. In this study, we fabricated a SERS substrate based on molybdenum oxide, using reactive DC magnetron sputtering and then used different simple and effective strategies to enhance the Raman signal. The results show that nanorod structure, oxygen deficiency engineering, phase engineering, and optical properties can be easily controlled by varying sputtering time and annealing time of MoOx SERS substrates. The analysis methods XRD, PL, and Raman show that with the optimal fabricated conditions. The presence of oxygen defects and a mixed MoO3, Mo9O26 phase structure in as well as the nanorod structure of MoOx SERS substrates could likely enhance Raman signals via a chemical mechanism (CM) and electromagnetic mechanism (EM). The MoOx SERS substrates were also used to detect R6G at low concentrations, with an EF of 1.14 × 106 (at 0.01 ppm), LOD of 0.01 ppm, and good temporal stability and reproducibility.