AbstractPolyvinyl alcohol (PVA)‐protected silver nanoarchitecture (PVAAg nanofilm) on the surface of the glass substrate was prepared by using electrostatic self‐assembly at a proper voltage. The two‐dimensional morphology of the PVAAg nanofilm has been examined by scanning electron microscopy (SEM). The surface‐enhanced Raman scattering (SERS) spectra of human serum (HS) on PVAAg nanofilms were recorded. The results show that the Raman scattering of HS can be enhanced efficiently based on these PVAAg nanofilms. However, it also can be seen that the effect of sodium citrate (SC) acting as anticoagulant on the SERS spectrum of HS is unnegligible, which has not been discussed adequately in the previous reports. To discuss the effect of SC on the SERS spectrum of HS, we have studied the normal Raman spectra of solid SC and the SERS spectra of 1.0 × 10−3 mol/l aqueous solution of SC adsorbed on the PVA–Ag nanofilms. Meanwhile, Raman wavenumbers of the SC molecule were calculated by using the method of DFT‐B3LYP/6‐31G*, and the dominant assignations of the calculated wavenumbers were performed. It was found that the density functional theory (DFT) calculation of SC Raman spectrum matches well with the experimental results. With the perfect reproducibility and high SERS activity, this method will be useful in the development of HS detection methods. Copyright © 2010 John Wiley & Sons, Ltd.