A flexible surface-enhanced Raman scattering (SERS) substrate with a nano-bowl array structure was constructed and used for the analysis and detection of Rhodamine 6G and thiram. First, a single-layer polystyrene (PS) microsphere film was prepared on a glass slide using the gas-liquid self-assembly method. Then, the singlelayer silver nanoparticles obtained by the liquid-liquid interface self-assembly method were transferred to the PS microsphere film. Subsequently, polydimethylsiloxane (PDMS) was coated on the silver nanoparticles. After drying and peeling, an AgNPs@ PDMS nanobowl array substrate was obtained. Using a portable Raman spectrometer to test this substrate with Rhodamine 6G as the probe molecule, the detection limit was found to be 1×10-8 mol/L, with an enhancement factor of 4.32×105. The test results also showed that the substrate has high uniformity and reusability. Using thiram as the probe molecule, the detection limit was as low as 1×10-6 mol/L. The relationship between the Raman peak intensity of thiram and its concentration was studied, and the results showed that the substrate can achieve quantitative detection of thiram.