The self-assembled ring like nickel (RnNi ∼ 10-20 nm) nanoparticles have been prepared by pulsed laser ablation method and confirmed by transmission electron microscopy. These RnNi nanoparticles electrophoretically deposited onto the indium-tin-oxide (ITO) glass substrate have been functionalized with dimethyl sulfoxide (DMSO) for covalent immobilization of anti-aflatoxin (a-AfB1) monoclonal antibodies and bovine serum albumin as blocking agent. The electrochemical response studies of a-AfB1/DMSO/RnNi-film/ITO bioelectrode reveal linearity as 5-100 ngdL−1, detection limit of 32.7 ngdL−1, sensitivity of 0.59 μA/ng dL−1, and shelf-life of 60 days. The low value (1.3 × 1014 molL−1) of association constant (Ka) shows high affinity towards aflatoxin.