As part of our project of developing a new IR-based immunosensor, we investigated the functionalization of gold substrates with thin organic films containing biotin ligands, A two-step procedure was developed consisting of the chemisorption of short amine-terminated organosulfur compounds, followed by their reaction at the solid liquid interface with an activated ester derivative of biotin. Covalent binding of biotin to these attachment layers was assessed by Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS) and X-ray photoelectron spectroscopy (XPS), The interaction of activated biotin with alcohol- and carboxylic acid-terminated monolayers was also investigated, and, as expected, no binding occurred, Moreover, mixed layers of short alcohol- and amine-terminated thiolates were successfully constructed at the gold surfaces and were shown to be the most efficient for the covalent binding of biotin thanks to the blocking effect of the thioalcohol, which prevented direct adsorption of biotin to the gold surface.