A simplemethod for the fabrication of stable and highly active surface enhanced Raman scattering (SERS) substrate by exploiting the optical properties of the silver nanoparticles (AgNPs) and organizational characteristics of the polymer is presented. Homogeneous distribution ofAgNPs is achieved with the usage of poly (4,7-di-2,3-dihydrothieno [3,4-b] [1,4] dioxin-5-yl-2,1,3 benzoselena diazole) (PESeE) film coated on the indium tin oxide glass (ITO) surface. The obtained structure ensured the emergence of a large number of hot spots where the localization of electromagnetic energy can result in enhancement of the Raman signal. The effect of the PESeE film thickness, the density of AgNPs added to the polymer film, and the concentration of silver ion solution on the morphology of the substrate and the enhancement of the SERS signal was revealed by using field emission scanning electron microscopy (FE-SEM) and SERS measurements. Enhancement power, homogeneity, and stability of the PESeE-AgNPs substrate were also investigated with measurement of the Raman probe. Spot-to-spot and batch-to-batch reproducibilities of the prepared substrate were calculated as 8.4%, and 10.2% (RSD %) respectively. Due to these properties, PESeE-AgNPs SERS substrate can be a good candidate for the detection and sensor application of various biological and chemical analytes. (C) 2019 The Electrochemical Society.