Surface-enhanced Raman detection of thiram is demonstrated by using Ag-nanoparticles (Ag NPs) on Au film over nanosphere (AuFON) substrate as the hybrid substrate. The SERS signal of the Ag NPs attached to solid supports is studied. The close coupling together of thousands of Ag NPs on AuFON leads to the generation of hot spots for SERS. The Ag NPs on AuFON can be applied to detect rhodamine-6G (R6G) with the detection limitation of 10⁻¹¹ M and the pesticide thiram in acetone with a detection limit of as low as 0.24 ppm, which is much lower than the maximal residue limit (MRL) of 7 ppm in fruit prescribed by the U.S. Environmental Protection Agency (EPA). The hybrid substrates are shown to be highly sensitive for the detection of thriam, which produce highly enhanced Raman signals with good uniformity and reproducibility due to having plenty of hot spots on its surface.