The residue of methyl parathion (MP) pesticide on agricultural products causes some health-related problems; therefore, it is necessary to identify the amount of this pesticide on these products. Surface Enhanced Raman Spectroscopy (SERS) is a fast, reliable, as well as accurate technique for detecting trace pesticides, biological substances, and chemicals. For this purpose, filter paper substrates (FPS) coated with gold nanoparticles (AuNPs) were used as SERS-active sensors to detect MP. First, AuNPs were fabricated by a simple chemical reduction method. Then, UV–Vis, XRD, TEM, FE-SEM, and DLS were employed to characterize the prepared AuNPs. Afterward, the AuNPs were coated dropwise on FPS to prepare flexible SERS-active substrates (Flex-SERS-Subs) to detect molecular vibrations of MP. The limit of detection (LOD) of detecting MP was obtained to be 10−12 M. The Raman spectra and peak signals acquired via experiments were validated using the density functional theory (DFT) method. Moreover, the RSD was calculated to be 3.39% for ten repeated measurements. In addition, the enhancement factor was calculated by experimental and numerical methods to be 9.820 × 104 and 10.324 × 104, respectively. Therefore, the developed method showed promising outcomes for the related investigations and has the potential to lead the development of similarly fabricated sensors for the detection of trace pesticides.