We demonstrate the fabrication of a label-free immunosensor for electrochemical detection of mycotoxin i.e. aflatoxin B-1 (AFB(1)) using nickel nanoparticle decorated reduced graphene oxide (rGO-Ni NPs) sheets. The rGO-Ni NPs have been electrophoretically deposited onto an indium tin oxide (ITO) coated glass electrode. This fabricated immunosensor is sensitive to 0.16 ng mL(-1) concentration of AFB(1) in phosphate buffer solution. It exhibits a lower limit of detection when compared to the HPLC standard method (2.5 ng mL(-1)) and shows minimal interference with another food toxin (ochratoxin A). Electron microscopy, Fourier transform infrared, UV-visible spectroscopy and electrochemical techniques were utilized to confirm selective decoration of Ni NPs on the rGO sheets and to investigate the chemical and electrochemical properties of the nanocomposite. The highly crystalline properties of the rGO-Ni NPs sheets along with the excellent electro-catalytic properties of the Ni NPs result in a higher heterogeneous electron transfer rate indicating the enhanced sensitivity (129.6 mA ng(-1) mL cm(-2)) of the device. The favorable environment for antibody conjugation on the surface of the rGO-Ni NPs sheets offers a low limit-of-detection (0.16 ng mL(-1)) for AFB(1)