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The continuous tuning of plasmonic nanoassembly’s structure is the key to manipulate their optical and catalytic properties. Herein, we report a strategy of using macroscopic deformation to continuously tune the structure and optical activity of massive plasmonic nanoassemblies that are embedded in elastic polymer matrix. Plasmonic gold nanoparticles (Au NPs) are assembled to nanochains (Au NCs) with defined length and further embedded into polyvinylpyrrolidone (PVP) matrix. The nanostructure and plasmonic properties of massive Au NCs in this Au NCs-PVP film can be simultaneously and continuously tuned, simply by reversible mechanical deformation of this elastic film. In this way, the surface-enhanced Raman scattering (SERS) enhancement factor of this film as a SERS substrate can be mechanically modulated in the range of 100 to 6.8 ×107. Meanwhile, the PVP matrix also serves as a selective diffusion barrier to eliminate the fluorescence interference of large biomolecules, which enables the Au NCs-PVP film as a convenient SERS substrate for quick and direct analysis of small molecule analytes in biological samples and food, avoiding the complicate and time-consuming sample pretreatment process.