Large-scale production and wide application of industrial dyes have considerably impacted the Earth’s ecology. Water pollution is very serious. Traditional chromatographic and spectral tools cannot detect weak spectral and chemical information. Therefore, the development of portable and rapid detection technology is essential. Surface-enhanced Raman spectroscopy (SERS) is a new analytical technique combined with nanotechnology, which can realize the detection of single molecular weight chemical substances. However, the potential is easily limited by the universality of SERS substrate enhancement ability and stability. In this study, a simple and general strategy was proposed to prepare a hydrophobic organic semiconductor bis (dicyanomethylene)-end-capped-dithieno ［2,3-d; 2’,3’-d］ benzo ［2,1-b; 3,4-b’］-dithiophene (4CN-DTmBDT) film is a new SERS substrate. Firstly, the organic semiconductor substrate was prepared by the spin-coating method. The π-conjugated organic semiconductor has the advantages of controllable molecular structure, biocompatibility, fine-tuning of photoelectric properties and controllable film-forming morphological parameters. The substrate surface has hydrophobicity, which makes silver nanoparticles (AgNPs) form a tight coffee ring on its surface. The organic semiconductor-nano silver SERS composite substrate was prepared to explore the enhancement effect of the Raman signal of the substrate. This study proposed a possible synergistic enhancement mechanism between the organic semiconductor and silver nanoparticles, and the enhancement ability and mechanism were studied. The results showed that the formation of tight coffee rings reduced the space between silver nanoparticles, and the hot spot effect was enhanced by concentrating the analyte. The detection limit of rhodamine 6G (R6G) with organic dye as the probe molecule was as low as 1×10-8 mol·L-1, and the SERS enhancement factor (EF) was as high as 1.30×106, while the detection limit of PDMS and silver nanoparticles composite substrate with better hydrophobicity was 1×10-5 mol·L-1. At the same time, it was proved that the substrate Raman signal was further enhanced by the synergistic effect between the organic semiconductor and silver nanoparticles in this study, the sensitivity was high, and the repeatability was good. The relative standard deviations (RSD) for detecting 1×10-4 and 1×10-8 mol·L-1 R6G dyes were 8.3% and 4.7%, respectively. Experiments show that the organic semiconductor-nano silver composite substrate has good application potential in trace analysis of dyes in wastewater.