Molecularly imprinted polymers (MIPs) embody a new class of materials possessing high selectivity and affinity for the target molecules. The aim of the present work is to explore the feasibility of employing the MIP as sensing material for the detection of Staphylococcal enterotoxin B (SEB) by Surface Plasmon Resonance (SPR). In this context, the MIP film was prepared by in-situ electropolymerization of 3-aminophenylboronicacid (3-APBA) on the bare gold chip in the presence of SEB molecule. The MIP of SEB exhibited a linear response from 3.2 fM to 25.6 fM (r2 = 0.99) with a detection limit of 0.05 fM. KD (equilibrium constant) and Bmax (maximum binding capacity of analyte) were calculated by using kinetic evaluation software and found to be 24 fM and 71, respectively and change in Gibb's free energy (G) value calculated using Vant's Hoff equation was found to be -77.54 kJ/mol. Interference study was performed with the homologs of SEB such as SEA and SEC in order to know the selectivity efficiency of SEB MIP and were found to be 23.57% and 23.43%, respectively. © 2010 Elsevier B.V. All rights reserved.