In the present work, a new substrate is proposed for the surface‐enhanced Raman spectroscopy (SERS) analysis of samples, which are of cultural heritage importance. A new and simple procedure is presented for the preparation of a stable SERS‐active substrate. It is based on the photoreduction of silver nitrate by ultraviolet light utilising hydroxypropyl cellulose as stabilising agent. The substrate's characteristics were tested and compared with a known substrate: a citrate‐reduced silver colloid, with alizarin as a reference material. Using the new substrate, it was possible to positively detect the organic dye alizarin red S, and two organic pigments: madder lake and alizarin crimson dark, as well as organic colourants prepared in paint layers with different organic binders without an interfering signal arising from the media, and without sample pre‐treatment. Furthermore, the investigated substrate also shows promising characteristics for the analysis of the cross sections of the samples because of its viscosity and the possibility of maintaining better control of the application of the substrate to the layer of interest. Copyright © 2014 John Wiley & Sons, Ltd. A new UV‐photoreduced surface‐enhanced Raman spectroscopy (SERS) substrate consisting of silver nanoparticles is proposed for the examination of works of art. Substrate enabled a successful detection of selected organic colourants (alizarin red S, madder lake and alizarin crimson dark), through a prominent enhancement of Raman signal, and fluorescence quenching. Furthermore, a selective analysis of organic colourants also in paint layers, without sample pre‐treatment, and a positive identification of an organic dye present in the glaze layer on a cross section of a sample were achieved.