This study presents an ultrasensitive electrochemical (EC) immunosensor for the detection of aflatoxin B1 (AFB1) using Nafion-immobilized functionalized multiwalled carbon nanotubes (MWCNTs). The Nafion film stabilizes the functionalized MWCNTs on the surface of an Au electrode, enhancing the sensor's performance. Functionalized MWCNTs, with their large specific surface area and numerous active sites, couple with anti-AFB1 monoclonal antibodies (mAbs), serving as a signal amplifier. In the presence of AFB1, it is specifically captured by the mAbs, reducing the amplified current signals, which are recorded by differential pulse voltammetry for accurate quantitation. The developed EC immunosensor exhibits high selectivity, excellent sensitivity with a limit of detection as low as 0.021 ng/mL, and a wide dynamic range of 0.05–100 ng/mL. It also shows good stability, reusability, and easy construction, making it suitable for real-world applications in food safety and environmental monitoring. The sensor's accuracy and practicality were confirmed by measuring AFB1 in fortified malt, lotus seed, and hirudo samples, with satisfactory recovery rates of 92.08–104.62%.This study presents an ultrasensitive electrochemical (EC) immunosensor for the detection of aflatoxin B1 (AFB1) using Nafion-immobilized functionalized multiwalled carbon nanotubes (MWCNTs). The Nafion film stabilizes the functionalized MWCNTs on the surface of an Au electrode, enhancing the sensor's performance. Functionalized MWCNTs, with their large specific surface area and numerous active sites, couple with anti-AFB1 monoclonal antibodies (mAbs), serving as a signal amplifier. In the presence of AFB1, it is specifically captured by the mAbs, reducing the amplified current signals, which are recorded by differential pulse voltammetry for accurate quantitation. The developed EC immunosensor exhibits high selectivity, excellent sensitivity with a limit of detection as low as 0.021 ng/mL, and a wide dynamic range of 0.05–100 ng/mL. It also shows good stability, reusability, and easy construction, making it suitable for real-world applications in food safety and environmental monitoring. The sensor's accuracy and practicality were confirmed by measuring AFB1 in fortified malt, lotus seed, and hirudo samples, with satisfactory recovery rates of 92.08–104.62%.