A flexible nanocomposite composed of bacterial cellulose (BC) and gold nanoparticles (AuNPs) was developed as a SERS substrate to determine thiram on apple surface by two collection methods namely “paste-and-peel” and “wiping”. Enhancement factor of this SERS substrate for sensing thiram residues was determined to be 2.8 × 105. Compared to the benchtop Raman spectrometer, portable Raman spectroscopic device showed a lower sensitivity towards thiram residues with limit of detection at 0.98 ppm, satisfying maximum residue level of thiram on apple required by both Europe and America. A good linear correlation of SERS peak intensity at 1368 cm−1 and different concentrations of thiram (1–50 ppm) revealed a coefficient up to 0.99. This flexible BC-based SERS substrate has a great analytical performance in sensitivity, reproducibility and stability, and is suitable for rapid detection (<8 min) and quantitative analysis of pesticides on food surface.