Researchers have struggled to develop reliable, reproducible, and cost-effective surface-enhanced Raman scattering (SERS) substrates for detecting compounds in complicated systems. In this study, a hydrophobic filter paper with an intrinsic internal standard (IS) SERS substrate was prepared. The sunflower-like Au and Prussian blue (PB) core-shell nanoparticles (NPs) were used as the basis for Raman signal enhancement and IS Raman signal to increase the reproducibility and sensitivity. Furthermore, the lubricant-treated Polytetrafluoroethylene (PTFE) filter papers featured a smooth interface as the support, which further improved uniformity, sensitivity and extended the application. Due to the distinctive design, the fabricated hydrophobic paper-based with IS SERS substrate showed impressive uniformity and sensitivity in the detection of R6G, paraquat and thiram, with the detection limit as low as 7.1 nM, 71 nM and 32 nM, respectively.  This substrate provided excellent uniformity and reproducibility with relative standard deviation (RSD) values of 9.46% and 10.26% of 15 points and 10 branches, respectively. In addition, this substrate was also validated successfully in Chinese herbal medicines (CHM), which contained multiple effective compounds. The satisfactory recovery results demonstrated that this substrate had a high potential to detect chemical contaminants in complex systems.