Surface-enhanced Raman scattering (SERS) has been widely used in food and drug detection, biological and medical sensing. In recent years, the study of non-metallic SERS substrates has gradually become a hot field of SERS. Here, we investigate the modulation effect on SERS activities of 2,3,5,6-tetrafluoro-7,7,8,8-tetrachyanoquindimethylene (F₄TCNQ) grown on molybdenum disulfide (MoS₂) films. The different nanostructures of F₄TCNQ can have an effect on the bound capability of charges transferred from the surface of MoS₂, which changes the electron density distribution on the surface of the F₄TCNQ/MoS₂ nanocomposite material. Therefore, the interface exhibits different charge localizations in the F₄TCNQ/MoS₂ nanocomposite. The charge transfer efficiency between the substrate and the adsorbed probe molecules leads the substrate to show a different SERS sensitivity. The enhancement factor of 4-mercaptobenzoic acid (4-MBA) molecules on the most optimized 7-min F₄TCNQ/MoS₂ nanocomposite substrate can reach 6.9\times 10^4 , and the detection limit concentration is as low as 10^–6 mol/L. The result of research on F₄TCNQ/MoS₂ nanocomposite provides an effective optimization scheme of energy level regulation for SERS based on the chemical enhancement mechanism, and opens up a new way to further exploit its functional applications.