The invention provides a gold nanosheet modified carbon fiber SERS substrate material, and a preparation method and an application thereof. The preparation method comprises the following steps: with carbon fibers as carriers, adsorbing TiO2 nanoparticles on the surfaces of the carbon fibers; placing the carbon fibers adsorbing the TiO2 nanoparticles on the surfaces in a mixed solution of hydrochloric acid and a titanium source to perform a hydrothermal reaction so as to grow TiO2 nano-column arrays on the surfaces of the carbon fibers; soaking the carbon fibers with the grown TiO2 nano-columnarrays on the surfaces in a solution containing gold ions to perform the hydrothermal reaction to deposit a gold nanosheet, cleaning the product with deionized water after the reaction, and drying theproduct to obtain the gold nanosheet modified carbon fiber SERS substrate material. The gold nanosheet modified carbon fiber SERS substrate material prepared by the preparation method provided by theinvention not only has relatively good SERS enhancement effect, but also has good uniformity and stability, and can be well applied to the analysis and detection of organic and biomolecules.