The present study demonstrated a one-step method for the first time to fabricate self-assembled gold nanoparticle (AuNP) metafilms at the water-toluene interface by adding polystyrene-polyisoprene-polystyrene as the support layer. The thiolated polyethylene glycol and ethanol were used to tune the surface charge density on the AuNPs, constructing a balanced situation at the water-toluene interface. The flexible (AuNP) metafilm can be easily obtained after evaporation of the toluene phase and further used as a surface-enhanced Raman scattering (SERS) substrate for trace thiram detection. The SERS sensitivity was tested using standard Raman probes such as crystal violet and malachite green, both with the detect concentration reaching 1 x 10(-11) M. Moreover, the excellent reproducibility and elastic properties make the metafilm promising in practical detection. Hence, the trace thiram detection on an orange pericarp was inspected with the detection limit of 0.5 ppm (1 x 10(-6) M) as well as a favorable linearity relation with a correlation coefficient of 0.979, exactly matching the realistic application requirements.