Herein, a sensitive and selective electrochemical sensor based on aptamer folding was constructed to detect aflatoxin B1 (AFB1) in peanuts. Specifically, polyethylenimine-functionalized multiwalled carbon nanotubes modified with molybdenum disulfide (MoS2@MWCNTs-PEI) were used as the electrode matrix to enable a large specific surface area, which were characterized by the Randles-Sevcik equation. Additionally, AuNPs were used to immobilize the aptamer via the Au-S covalent bond and provide a favorable microenvironment for signal enhancement. Methylene blue (MB) was modified at the proximal 3' termini of the aptamer as the capture probe, while the signal transduction of the sensor was obtained through changes in conformation and position of MB induced by the binding between AFB1 and the probe. Changes in spatial conformation could be recorded by electrochemical methods more readily. This electrochemical aptasensor demonstrated remarkable sensitivity to AFB1 with an extensive detection range (1 pg/mL to 100 ng/mL) and a lower limit detection (1.0 × 10-3 ng/mL). Moreover, using the constructed aptasensor, AFB1 was identified successfully in peanut samples, with recoveries ranging from 95.83 to 107.53%, illustrating its potential use in determining AFB1 in food.