A label-free biosensor based on surface plasmon resonance (SPR) has been developed to evaluate the potential of peptide ligands as biorecognition molecules for the detection of bacterial toxins. A 24-mer peptide, previously identified from a phage display library and modified for better binding, was used for the detection of staphylococcal enterotoxin B (SEB). The peptide was immobilized covalently onto the gold-coated surface modified with self-assembly monolayer. The limit of detection (LOD) for SEB was found to be 20 mu g/mL. The selectivity of the peptide-based sensor for SEB was verified by injecting bovine serum albumin (BSA), and the sensor response for the BSA was negligible. Further, to probe the effectiveness of the sensor in complex food environments, it was tested with milk samples spiked with SEB, showing that it is applicable to complex food matrices. The results of this study demonstrate the potential of peptides, selected from phage display libraries, as recognition agents for detecting bacterial toxins using biosensors.