In this study, an ultrasensitive electrochemical (EC) aptasensor with Nafion-stabilized functionalized multi-walled carbon nanotubes (f-MWCNTs) as signal enhancers was established for ochratoxin A (OTA) determination. Herein, f-MWCNTs were prepared through functionalization with nitric acid. The incorporation of Nafion promoted a good dispersion of f-MWCNTs and prevented their leaching on the electrode, making a robust stability of the aptasensor. The Nafion-f-MWCNTs composites were used as the sensing substrates to largely enhance the electroactive surface area and the conductivity of the electrode, realizing a significant signal amplification. Carboxyl groups on the surface of f-MWCNTs readily exposed from Nafion membrane to couple with streptavidin, facilitating the immobilization of biotinylated aptamers to achieve selective recognition towards OTA. When OTA existed, aptamers preferentially combined with it, causing a noticeable decline in the current response. Under optimum conditions, a good linear relationship between the current changes and the logarithm of OTA concentration was observed from 0.005 ng/mL to 10 ng/mL, with a limit of detection low to 1 pg/mL for OTA. The specific, sensitive, and reproducible aptasensor succeeded in application in malt samples, confirming a great promise for more contaminants and providing a universal platform in complex matrices by simply replacing the corresponding aptamers.