The ubiquitous aflatoxin B 1 (AFB 1 ) contamination in foods and other complex matrices has brought great challenges for onsite monitoring. In this study, an ultrasensitive Nafion-immobilized functionalized multiwalled carbon nanotube (MWCNT)-based electrochemical (EC) immunosensor was developed for trace AFB 1 detection. The introduced Nafion film could steadily stabilize functionalized MWCNTs with uniform distribution and tiling on the surface of a Au electrode. Functionalized MWCNTs with a large specific surface area, numerous active sites to couple with abundant anti-AFB 1 monoclonal antibodies (mAbs), and high conductivity served as the signal amplifier for remarkably enhancing the sensing performance of the immunosensor. In the presence of AFB 1 , it was specifically captured by mAbs to reduce the amplified current signals, which were recorded by differential pulse voltammetry for the accurate quantitation of AFB 1 . Because of the synergistic effects of Nafion on the stabilization of functionalized MWCNTs as signal enhancers, the developed EC immunosensor exhibited an extremely 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, besides fascinating merits of easy construction, low cost, good stability in 7 days, and good reusability. The anti-interference ability of the immunosensor was verified against three other mycotoxins, and the practicability and accuracy were confirmed by measuring AFB 1 in fortified malt, lotus seed, and hirudo samples with a satisfactory recovery of 92.08−104.62%. This novel immunosensing platform could be extended to detect more mycotoxins in complex matrices to ensure food safety.