In this paper we report detailed Fourier transform infra-red (FTIR) and Raman spectroscopic results obtained after the modification of a porous silica surface using different steps of functionalization and protein grafting. After the elaboration of porous silicon (PS) layers, with sufficient adjusted pore size and well defined porosity, we carried out complete thermal oxidation and we optimized the silanization step by varying the concentration of 3-aminopropyltriethoxysilane (APTES) molecules using different immersion and rinsing durations. Then we introduced a coupling agent, glutaraldehyde (GL) molecules, which has a great affinity for bovine serum albumin (BSA) molecule grafting. FTIR and Raman spectroscopic analyses present complementary spectra that allow us to get an idea about the chemical links and vibrational modes that appear during the functionalization process and after protein attachment. This proves that the biological molecules are covalently attached on the modified porous silica surface. Moreover, a modelling study of the reflectance spectra allows values of the volume fraction and refractive index variations to be estimated by assigning them with the number of APTES, glutaraldehyde and protein layers after each step. These results are well correlated to those obtained by the FTIR and Raman analyses. This work on porous silica single-layers is carried out in order to exploit it for the elaboration of functionalized optical waveguides to obtain a sensitive label-free optical biosensor. (C) 2011 Elsevier B.V. All rights reserved.