Immobilisation of rabbit immunoglobulin G (rIgG) was performed by affinity binding to protein A (PrA) covalently bound to threedifferent thiolate self-assembled monolayers (SAMs), (i) a mixed SAM of mercaptoundecanoic acid (MUA) and mercaptohexanol(C6OH) at a molar ratio of 1–3, (ii) a pure SAM of MUA and (iii) a pure SAM of cystamine (CA). A comparative study of anti-rIgGrecognition process on these three surfaces was achieved in order to assess the influence of the attachment layer topography and compositionupon the sensor quality. Functionalised gold-coated surfaces were characterised by three complementary analytical techniques,namely atomic force microscopy (AFM), polarization modulation–reflection-adsorption infrared spectroscopy (PM-RAIRS) and quartzcrystal microbalance (QCM). PM-RAIRS and AFM revealed that the three SAMs were formed on the gold surfaces. AFM observationsmade it clear that the thiolate and PrA layers were rather homogeneous in the case of pure MUA and CA SAMs, as compared to theMUA/C6OH mixed SAM on which PrA aggregates were observed. Though the highest amount of antibody was bound to the PrA onCA layer, higher anti-rIgG over IgG ratios were measured on the less dense layers of antibody.