This article developed a novel electrochemical immunosensor for the specific detection of aflatoxin B1 (AFB1). Amino‐functionalized iron oxide nanoparticles (Fe3O4‐NH2) were synthesized. Fe3O4‐NH2 were chemically bound on self‐assembly monolayers (SAMs) of mercaptobenzoic acid (MBA). Finally, polyclonal antibodies (pAb) were immobilized on Fe3O4‐NH2‐MBA. The sensor system was evaluated through atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). A reduction in the anodic and cathodic peak currents was observed after the assembly of the sensor platform. The charge transfer resistance (Rct) was increased due to the electrically insulating bioconjugates. Then, the specific interaction between the sensor platform and AFB1 blocks the electron transfer of the [Fe(CN)6]3−/4− redox pair. The nanoimmunosensor showed a linear response range estimated from 0.5 to 30 μg/mL with a limit of detection (LOD) of 9.47 μg/mL and a limit of quantification (LOQ) of 28.72 μg/mL for AFB1 identification in a purified sample. In addition, a LOD of 3.79 μg/mL, a LOQ of 11.48 μg/mL, and a regression coefficient of 0.9891 were estimated for biodetection tests on peanut samples. The proposed immunosensor represents a simple alternative, successfully applied in detecting AFB1 in peanuts, and therefore, represents a valuable tool for ensuring food safety.