We describe the elaboration of ultra-sensitive immunosensors, to detect the bacterial pathogen Staphylococcus aureus. We utilized commercially available polyclonal anti-S. aureus antibody as receptor. Immunosensors were elaborated by building a self-assembled monolayer (SAM) of thiolamine onto planar gold-coated sensor chips. Then, Protein A was covalently linked to the thiolated SAM using glutaraldehyde as cross-linking agent. After a blocking step by Bovine Serum Albumin (BSA), the antibody was immobilized by affinity to Protein A. This step-by-step construction was monitored by Polarization Modulation Reflection Absorption Infrared Spectroscopy (PM-RAIRS) and Quartz Crystal Microbalance with Dissipation (QCM-D). In a first stage, the parameters of immunosensor elaboration were optimized using a model rabbit IgG. The accessibility of receptors and the homogeneity of their distribution were checked by PM-RAIRS, QCM-D, and by immuno-gold scanning electron microscopy. Then, the specific rabbit anti-S. aureus antibody was immobilized and the resulting sensing layer was applied to the detection of the pathogen target. Independent detection of bacteria immobilized on the sensors by fluorescent imaging allowed validation of the specificity of recognition toward the pathogen as well as a quantitative response of the sensor. Using PM-RAIRS as transducing technique allowed us to enhance sensitivity and reach a very competitive detection level (10(5) CFU mL(-1)).