An immunosensor with quartz crystal microbalance (QCM) detection operating in flow-through mode was set up for assaying staphylococcal enterotoxin A (SEA) in model buffer medium and in milk. This biosensor is label-free and operates in the direct format. Each step of construction of the sensing layer comprising the capture anti-SEA antibody was investigated at the molecular level and optimized. The molecular layer was built using either amine- or acid-terminated thiols, with and without adding protein A to immobilize anti-SEA. The most efficient strategy to immobilize the antibody was selected on the basis of the biosensor's response to a standard solution of SEA. The optimized sensing layer was successfully used for the direct and fast (15 min) assay of SEA in phosphate buffer by QCM within a working range of 0.05-1 mg L−1 and a limit of detection of 0.02 mg L−1. Using a sandwich type assay, the response was amplified by a factor of 2 and consequently the lowest measurable concentration dropped down to 0.007 mg L−1 for a total assay time of 25 min. Furthermore, sensor regeneration was achieved in good conditions with low pH buffer releasing solution. Two levels of regeneration were reached, the first one, up to the binding protein level, necessitating the rebinding of anti-SEA. In this case the response of the regenerated sensor was 73% in the first cycle then stabilized at 60% of the primary response. For the second level, the capture antibody was cross-linked to the underlayer of protein A and no further grafting was needed. In this second case, the response of the regenerated sensor was 65% of the primary response. Eventually, the piezoelectric immunosensor was successfully applied to the direct assay of SEA spiked in real milk samples.