Flexible surface-enhanced Raman spectroscopy (SERS) substrate has attracted great attention due to its convenient sampling and on-site monitoring capability. However, it is still challenging to fabricate a versatile flexible SERS substrate, which can be used for in situ detection of analytes either in water or on irregular solid surfaces. Here, we report a flexible and transparent SERS substrate based on a wrinkled polydimethylsiloxane (PDMS) film obtained by transferring corrugated structures on the aluminium/polystyrene bilayer film, onto which silver nanoparticles (Ag NPs) are deposited by thermal evaporation. The as-fabricated SERS substrate exhibits a high enhancement factor (∼1.19×105), good signal uniformity (RSD of 6.27%), and excellent batch-to-batch reproducibility (RSD of 7.3%) for rhodamine 6 G. In addition, the Ag NPs@W-PDMS film can maintain high detection sensitivity even after mechanical deformations of bending or torsion for 100 cycles. More importantly, being flexible, transparent, and light, the Ag NPs@W-PDMS film can both float on the water surface and conformally contact with the curved surface for in situ detection. The malachite green in aqueous environment and on apple peel can be easily detected down to 10-6 M with a portable Raman spectrometer. Therefore, it is expected that such a versatile flexible SERS substrate has great potential in on-site, in situ contaminant monitoring for realistic applications.