We propose a simple, low-cost, large-area, and functional surface enhanced Raman scattering (SERS) substrate for biomedical applications. The SERS substrate with chitosan-silver nanoparticles (chitosan-Ag NPs) hybrid 3D porous structure was fabricated simply by a one-step method. The chitosan was used as a template for the Ag NPs deposition. SERS enhancement by the chitosan-Ag NPs substrate was experimentally verified using rhodamine B as an analyte. Thiolated single stranded DNA was also measured for atopic dermatitis genetic markers (chemokines CCL17) at a low concentration of 5 pM. We successfully designed a novel SERS substrate with silver nanoparticle hybridized 3D porous chitosan that has the potential to become a highly sensitive and selective tool for biomedical applications. (C) 2013 Elsevier B.V. All rights reserved.