Surface Enhanced Raman Spectroscopy (SERS) is one of the most sensitive analytical techniques currently available and has been widely used in life science, material science, environmental science and analytical chemistry. The performance of SERS base determines the application scope of SERS, are the key to promoting the development of SERS technology, the preparation of highly active SERS substrate has become a hot spot in the SERS research field. In order to obtain the best Raman signal, SERS active substrate with special characteristics is great demanded. Flexible SERS substrate has unique advantages such as good flexibility, 3D scaffold structure and controllable pore size on the surface, and it is a good application value in detecting compounds and bacteria. The surface of Nylon flexible film has the characteristics of the hierarchical and porous staggered structure. Au-Nylon flexible film substrate with high SERS activity was prepared by combining the solid phase extraction unit with the special material Nylon by changing the amount of gold sol and binding times of gold nanoparticles and film. The result showed that gold nanoparticles attach well to Nylon fibers, and the plasmon resonance absorption peak of the Au-Nylon flexible film substrate has blue moved, which was due to the intrinsic surface plasmon resonance coupling of gold and Nylon, forming Au nanoparticles and Nylon fiber complex. After the first treatment, Nylon fiber and its attached Au nanoparticles formed a new active retention layer, which contributes to making the gold particles better attached to the film’s surface during the second treatment, forming SERS “hot spot” effect and improving its SERS performance. The SERS performance of the Au-Nylon flexible film substrate was analyzed by using Crystal Violet (CV) as the SERS probe molecule. It was found that the SERS strength of the CV probe molecules on the Au-Nylon flexible film substrate varied as the number of filtration times and binding times of gold nanoparticles and film. For Au-Nylon flexible film substrate with an area of 1 cm², When a single gold sol amount of 1 mL is combined with the film two times, and the total binding amount is 2 mL, the SERS signal of the CV probe molecules and the activity of SERS samples was the highest. The Au-Nylon flexible film substrate was used for SERS detection of the CV solution with a concentration of 2.5×10^-5, 1×10^-5, 1×10^-6, 5×10^-7 and 1×10^-7 mol·L^-1. Respectively, The Au-Nylon flexible film substrate enhancement factor reached 1.0×10⁴, and the detection limit was 1.0×10^-6 mol·L^-1. In addition, The Au-Nylon flexible film substrate had good uniformity with a relative average deviation of 11.8%. The Au-Nylon film substrate still had good SERS performance in microbial detection, and the SERS enhanced effect on Staphylococcus aureus was better than that of gold sol. It could be seen that the Au-Nylon flexible film substrate has good homogeneity and good SERS activity. The method has the advantages of simple and easy batch preparation, and it has good practical application value in both compound detection and microbial detection.