In this work, we designed and fabricated a simple and convenient Au nanoparticle/graphene/Au nanoparticle (AuNP/G/AuNP) flexible ‘sandwich’ substrate on the surface of the polyethylene (PE) film through self-assembly. The coupling effect between the AuNPs enables this substrate to strongly enhance the local electromagnetic field, and graphene can also provide additional chemical improvement. Experimental results show that the low Rh6G concentration (10⁻⁹ M) located on the surface of the AuNP/G/AuNP substrate can be detected. Furthermore, the flexible and transparent AuNP/G/AuNP substrate can detect low concentrations of thiram (0.24 ppm) on the surface of orange peels. This low concentration is below the maximum residue limit (5 ppm) regulated by the National Standard of China in fruits and vegetables. Therefore, this flexible substrate can be used as an effective surface-enhanced Raman scattering sensor for in situ detection in food safety and environmental monitoring.