A novel electrochemical immunosensor based on Nafion-immobilized functionalized multiwalled carbon nanotubes (MWCNTs) was developed for the ultrasensitive detection of aflatoxin B1 (AFB1) in complex matrices. The sensor utilizes functionalized MWCNTs with a large surface area, numerous active sites, and high conductivity to amplify signals, while Nafion stabilizes the MWCNTs on the gold electrode surface. The sensor employs anti-AFB1 monoclonal antibodies (mAbs) for specific binding of AFB1, which reduces current signals recorded by differential pulse voltammetry (DPV) and cyclic voltammetry (CV). The sensor demonstrated high selectivity, excellent sensitivity with a detection limit of 0.021 ng/mL, a wide dynamic range of 0.05–100 ng/mL, and good stability and reusability. It showed high accuracy and recovery rates (92.08–104.62%) when analyzing AFB1 in fortified samples of malt, lotus seed, and hirudo. The sensor's performance was validated against three other mycotoxins, confirming its strong anti-interference ability. The developed immunosensor offers a promising platform for detecting AFB1 and other mycotoxins in complex food matrices, ensuring food safety. The sensor's simplicity, low cost, and environmental friendliness make it suitable for on-site and real-time monitoring of AFB1 in various samples.A novel electrochemical immunosensor based on Nafion-immobilized functionalized multiwalled carbon nanotubes (MWCNTs) was developed for the ultrasensitive detection of aflatoxin B1 (AFB1) in complex matrices. The sensor utilizes functionalized MWCNTs with a large surface area, numerous active sites, and high conductivity to amplify signals, while Nafion stabilizes the MWCNTs on the gold electrode surface. The sensor employs anti-AFB1 monoclonal antibodies (mAbs) for specific binding of AFB1, which reduces current signals recorded by differential pulse voltammetry (DPV) and cyclic voltammetry (CV). The sensor demonstrated high selectivity, excellent sensitivity with a detection limit of 0.021 ng/mL, a wide dynamic range of 0.05–100 ng/mL, and good stability and reusability. It showed high accuracy and recovery rates (92.08–104.62%) when analyzing AFB1 in fortified samples of malt, lotus seed, and hirudo. The sensor's performance was validated against three other mycotoxins, confirming its strong anti-interference ability. The developed immunosensor offers a promising platform for detecting AFB1 and other mycotoxins in complex food matrices, ensuring food safety. The sensor's simplicity, low cost, and environmental friendliness make it suitable for on-site and real-time monitoring of AFB1 in various samples.