This study describes the fabrication of an aflatoxin (AFB1) electrochemical immunosensor by immobilization of an anti-AFB1 antibody-bovine serum albumin (BSA) conjugate on a layer of L-cysteine (L-Cys) used to link the antibody to the palladium nanoparticle-boron nitride (PdNP-BN)-modified carbon felt (CF) electrode. Each step of the immunosensor fabrication was characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). These two analyses demonstrated that BN acted as a substrate for PdNPs and also as an electroactive material that enhanced the immunosensor electrical conductivity. Different features (antibody concentration, pH, incubation time) were optimized to enhance the immunosensor analytical performance. In these conditions, the fabricated immunosensor displayed a large linear range (1-10 ng mL-1 ; R 2 = 0.9987), low limit of detection (0.834 ng mL-1 AFB1), and was stable for up to 3 weeks. The fabricated electrochemical immunosensor could detect AFB1 in wine samples with recovery levels of 93-106%. Lastly, the Monte Carlo simulation studies confirmed the stronger binding of the anti-AFB1 antibody-BSA conjugate to the L-Cys/PdNP-BN/CF electrode surface, and molecular docking confirmed the docking of BSA subdomain II to the anti-AFB1 antibody.