A simple, reliable and reproducible one-step electrochemical method for the preparation of surface-enhanced Raman-active polymer-mediated silver nanoparticles (Ag NPs) on planar indium tin oxide (ITO) coated glass substrates was reported. Poly(3,4-ethylenedioxythiophene) (PEDOT) film was used as a support material for dispersing nanostructured silver nanostructures on the surface homogeneously, since 3,4-ethylenedioxythiophene (EDOT) monomer polymerizes regioregularly. The optical properties and morphologies of the silver substrates have been investigated by ultraviolet-visible (UV-vis) spectroscopy and field emission scanning electron microscopy (FE-SEM). The UV-vis and FE-SEM results revealed that the Ag nanostructures separately appeared on the PEDOT coated ITO after reduction. The effect of the thickness of PEDOT polymer film, reduction potential of silver, the concentration of silver ion solution and the amount of silver particle on the polymer film on the SERS response were studied as well as repeatability and temporal stability of prepared substrates. Brilliant cresyl blue (BCB) has been used as Raman probes to evaluate the properties of the new SERS substrates. Signals collected over multiple spots within the same substrate resulted in a relative standard deviation (RSD) of 9.34%, while an RSD of 11.05% was measured in signals collected from different substrates. The SERS-active substrates were robust and stable which lost only 5.71% of initial intensity after 1 month. (C) 2012 Elsevier Ltd. All rights reserved.