An immunosensor for determining staphylococcal enterotoxin A (SEA) was developed on the basis of surface plasmon resonance (SPR). The sensor surfaces were constructed from various thiol mixtures of different molar ratios of 16-mercapto-1-hexanol (16-MHA) to 6-mercapto-1-hexanol (6-MCH). The surface functionalized with a 1:20 mixed self-assembled monolayer(SAM) exhibited the best result in terms of SEA detection sensitivity. Through optimization of anti-SEA immobilization, the SPR sensor could detect SEA in phosphate-buffered saline buffer in a linear range from 100 to 1000ng/ml. Furthermore, studies of recovery and matrix effects were performed to evaluate the suitability of the developed immunosensor for directly analyzing SEA in milk samples. Results indicated that the mixed SAM-based SPR immunosensor has the potential for the specific detection of SEA. (C) 2008 Elsevier B.V. All rights reserved.