At present, the use of efficient and cost-effective methods to construct plasmonic surface-enhanced Raman scattering (SERS) substrates of high sensitivity, uniformity and reproducibility is still crucial to satisfy the practical application of SERS technology. In this paper, a localized surface plasmonic resonance (LSPR) tunable flexible Ag@PDMS substrate was successfully constructed by the low-cost bio-template-stripping method and magnetron sputtering technology. The theory proves that the local electromagnetic field enhancement and “hot spot” distribution is adjustable by modifying the size of the optical cavity unit in the periodicity nanocavity array structure. Experimentally, using rhodamine 6G (R6G) as the target analyte, the SERS performance of optimal Ag@PDMS substrate (Ag film thickness for 315 nm) was researched in detail, which the minimum detection limit was 10−11 M and the enhancement factor was calculated as 8.03 × 108, indicating its high sensitivity. The relative standard deviation (RSD) was calculated as 10.38%, showing that the prepared substrate had excellent electromagnetic field enhancement uniformity. At last, the trace detection of Crystal violet (CV, LOD = 10−9 M) and the simultaneous detection of three common dyes (R6G, CV and Methylene blue (MB) mixture) were also realized. This result suggests that the SERS substrate has a good application prospect in the quantitative and qualitative detection of dye molecules.