A label free impedimetric immunosensor for the determination of aflatoxin B1 (AFB1) was fabricated by immobilizing anti-AFB1 onto Poly o-phenylenediamine (PoPD) electropolymerized film modified gold electrode by glutaraldehyde (GA) cross-linking. An electrochemical interfacial modeling of biomolecular recognition was recommended and reasonably interpreted. EIS technology was employed for the quantitative determination. The linear detection concentration ranges of AFB1 were 0.03~0.1 μg/mL and 0.1~0.7 μg/mL. The detection limit was 2.8×10-8μg/mL. The immunosensor could be reused more than 10 times when renewed by HCl-Glycine buffer solution (pH 2.6).