We report results of the studies relating to the fabrication of a highly sensitive label free biosensor based on graphene oxide (GO) platform for the detection of aflatoxin B1 (AFB1) which is most toxic and predominant food toxin, using electrochemical impedance spectroscopy. The structural and optical characterization of GO/Au and anti-AFB1/GO/Au has been done by electron microscopy, Raman, X-ray diffraction (XRD), UV–vis and electrochemical impedance spectroscopy (EIS). The impedimetric sensing response of immunoelectrode as a function of AFB1 concentration reveals wider linear detection range (0.5–5 ng/ml), high sensitivity (639 Ω ng−1 ml), improved detection limit (0.23 ng ml−1) and good stability (5 weeks) for the label-free detection. Association constant (ka) for antigen–antibody interaction obtained as 0.46 ng ml−1 indicates high affinity.