In this study, we developed a novel reduced graphene oxide/molybdenum disulfide/polyaniline@gold nanoparticles-based electrochemical aptasensor (termed as RGO/MoS2/PANI@AuNPs/Apt) for detection of aflatoxin B1 (AFB1). The RGO/MoS2/PANI nanocomposites were synthesized and characterized by multiple techniques, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), infrared spectroscopy (FTIR), UV-visible spectroscopy, and X-ray photoelectron spectroscopy (XPS). A glassy carbon electrode (GCE) was then modified by the RGO/MoS2/PANI nanocomposites, coated with a chitosan (Cs) film, and followed by AuNPs attachment for immobilizing the AFB1 aptamers. In the presence of AFB1, the AFB1 binding-induced conformation change of the immobilized aptamer on the electrode surface results in the reduction of the electron transfer from a [Fe(CN)6]3-/4- redox couple in the solution to the GCE surface. Therefore, the aptamer-AFB1 binding event can be easily monitored by the peak current change of the RGO/MoS2/PANI@AuNPs/Apt through differential pulse voltammetry (DPV) measurement. Under the optimized conditions, the as-developed RGO/MoS2/PANI@AuNPs/Apt exhibits a wide linear range from 0.01 fg mL-1 to 1.0 fg mL-1 and a remarkably low detection limit (3σ) of 0.002 fg mL-1. The aptasensor also has good reproducibility as well as shows high selectivity against other fungal toxins, such as OTA and FB1. Moreover, the practicability of the RGO/MoS2/PANI@AuNPs/Apt was demonstrated by the analysis of AFB1 in the spiked wine samples.