We developed a novel method to fabricate uniformly distributed silver nanoparticles (AgNPs) on nanostructured polymer for use as surface-enhanced Raman spectroscopy (SERS) substrates. Over 92% reproducibility on the same substrate was obtained in SERS analyses of benzenethiol (BT) using this novel substrate. The reproducibility of the BT SERS spectra collected from different substrates is >82%. The new SERS substrates were further used to quantitatively analyze label-free dipeptide in aqueous solution. The Raman signal is reproducible with levels above 91% for solution concentrations from 1 mu g ml(-1) to 1 ng ml(-1) of the label-free dipeptide. The accurate and reproducible identification over a wide concentration range demonstrated that the new SERS substrate is suited for use in quantitative identification of biomolecules. (C) 2010 Elsevier B.V. All rights reserved.