In this study, an efficient, homogeneous, flexible and transparent Ag/Polydimethylsiloxane (PDMS) substrate with an orderly three-dimensional nanopillar structure was proposed. Ag NPs were thermally deposited on the flexible bionic PDMS support obtained by a two-step replication of cicada wings. Scanning electron microscope images reveal when the deposition time was 50 min, Ag NPs of proper size densely covered the entire PDMS nanopillar surface in the Ag-50/PDMS substrate. Both the gaps between the Ag NPs and those between the nanopillars acted as efficient electric field amplifiers. The enhancement factor (EF) of the Ag-50/PDMS substrate was calculated to be 2.89 × 107 by using crystal violet as the probe molecule. The Ag-50/PDMS substrate also exhibited good uniformity and reproducibility with a relative standard deviation of 1.46% and 11.45% respectively. The detection capability of the proposed flexible and transparent Ag-50/PDMS substrate in practical applications was demonstrated by the in-situ detection of 0.1 ppm malachite green on fish surfaces, indicating its great potential in the field of food monitoring.