In this work, the design of an impedimetric immunosensor for real-time detection of trace concentrations of Staphylococcus enterotoxin B (SEB) is presented. The nano-porous aluminum has been used as an immobilization matrix for the anti-SEB antibody. Using a 3-aminopropyltriethoxysilane as a coupling reagent allows to obtain a highly reproducible and stable sensing surface with significantly increased surface area. Variable potential electrochemical impedance spectroscopy has been used to establish optimal detection window and determine kinetic parameters of immunoreaction. Using developed immobilization method and optimized detection regime, it is possible to determine the presence of SEB at levels as low as 10 pg/ml. Differential measuring schemes allowed to separate the effects of the sample matrix and immunoreaction in the sensor's output signal.