In this paper we report a study of novel thermally stable surface enhanced Raman scattering (SERS) substrates consisting of Ag nanoparticles immobilized on an anodized aluminum oxide (AAO) support. The morphological and chemical characteristics and the SERS activity of the Ag nanoparticles before and after the thermal treatment were evaluated using SEM, XPS, UV-vis and Raman spectroscopy. Our results show that the nanoporous surface of AAO significantly hinders the fusion of Ag nanoparticles to single large particles at up to 400 °C, preserving high SERS enhancement. XPS and SERS results indicate that exposure to high temperatures efficiently ‘cleanses’ the surface from citrate remnants, opening more binding sites for analytes on Ag nanoparticles. In addition, thermal decomposition of silver oxide occurs at 400 °C, ensuring a pure metallic surface and further enhancing SERS activity.