We demonstrate a one-step synthesis and controlled assembly of Au nanoparticles (NPs) on lambda-DNA scaffolds by an in-situ photoreduction method. The interparticle gaps of the Au NPs attached on DNA can be fine regulated by controlling the R values (denoted as the ratio of Au(III) ions added per DNA base pair). At R = 20, the interparticle gap between two adjacent Au NPs on DNA is in the range of 1-3 nm para-aminothiophenol (PATP) as a Raman reporter is used to evaluate the surface-enhanced Raman scattering (SERS) performance of these DNA-Au hybrid substrates. Empirical enhancement factor above 1 x 10(9) is observed when the R value is in a fairly broad range of 10-60 under 785 nm excitation. At R = 20, this DNA-Au hybrid as SERS substrate can give a low detection limit of 0.5 nM of PATP, moreover, these substrates have a good reproducibility at different sites on a substrate, with a standard deviation of <15%. These results indicate that the photoreduced Au NPs on DNA scaffolds can be used as SERS-active substrates which exhibit high and reproducible SERS activity. This DNA-Au hybrid has potential applications in chemical and biological SERS analysis. Furthermore, the synthesis process is so simple and quick, which needs just only 15 min. Combined with the ability to make preformed scaffolds, the selective deposition of Au NPs on fixed DNA scaffolds will be an important addition to the rapidly growing nanofabrication tool kit for in-field SERS detection. (C) 2012 Elsevier B.V. All rights reserved.