A flexible and highly sensitive Surface-Enhanced Raman Scattering (SERS) film with PMMA/Ag/graphene/Ag/graphene hybrid structure was designed and prepared, which showed about a 2-fold enhancement in Raman signals of R6G molecules, compared with PMMA/Ag nanoparticles/graphene substrate. The SERS substrate provides very high sensitivity to R6G molecules reaching a detection limit of 1×10−8M. The substrate can be attached directly on the tested sample owing to its excellent flexibility. In the SERS test on the malachite green (MG) soaked fish samples, the use of such substrate makes the in-situ detection being accomplished within 5min under sensitivity of 10−7M, which shows very high practical utility.