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Abstract In this study, we develop a facile method to fabricate highly sensitive and stable surface-enhanced Raman scattering (SERS) substrate, which is realized by combining co-sputtering with atomic layer deposition technology. To accomplish the SERS substrate preparation, we firstly utilized co-sputtering silver and aluminum on glass slides to form uniform discontinuous Ag film by removing Al later, which acted as SERS active moiety and presented high sensitivity in glycerin detection. After coating an ultrathin TiO2 layer via atomic layer deposition (ALD), the samples could further enhance the Raman signal due to the chemical effect as well as the long-range effect of the enhanced electromagnetic field generated by the encapsulated Ag nanoparticles (NPs). Besides, the coated sample could maintain the significant enhancement in air condition for more than 30 days. The high stability is induced by TiO2 layer, which efficiently prevents Ag NPs from surface oxidation. This highly sensitive and stable SERS substrate might highlight the application of interface state investigation for exploring novel liquid lubricating materials.