The aim of this work is to demonstrate that hydroxocobalamin molecules (vitamin B12) may be grafted on functionalized fresh mesoporous silicon layers. A chemical process is proposed to functionalize and stabilize freshly prepared porous silicon (PS) substrates. The procedure consists of a direct silanization via hydrolyzed 3-aminopropyltriethoxysilane (APTES) of fresh PS layers and their functionalization by glutaraldehyde linker to bind vitamin B12 (VB12). We pointed out the formation of a silane layer on freshly PS substrates. The chemical surface modification of APTES-functionalized PS layers (APTES-PS) was investigated by Attenuated Total Reflectance Fourier Transform Infrared spectroscopy. Electrochemical Impedance Spectroscopy (EIS) measurements were performed to study the impedance changes following functionalization of the PS layer and grafting of VB12. EIS results indicate that VB12 is successfully immobilized on functionalized PS. Herein, we report for the first time the immobilization of vitamin B12 on functionalized PS layers. This study provides further evidence for the successful use of PS as a matrix for immobilization of biological molecules.