The increasing incidence of diseases caused by highly carcinogenic aflatoxin M1(AFM1) in food demands a simple, fast, and cost-effective detection technique capable of sensitively monitoring AFM1. Recent works predominantly focus on the electrochemical aptamer-based biosensor, which still faces challenges and high costs in experimentally identifying an efficient candidate aptamer. However, the direct electrochemical detection of AFM1has been scarcely reported thus far. In this study, we observed a significant influence on the electrochemical signals of ferric ions at a gold nanoparticle-modified glassy carbon electrode (AuNPs/GCE) by adding varying amounts of AFM1. Utilizing ferricyanide as a sensitive indicator of AFM1, we have introduced a novel approach for detecting AFM1, achieving an unprecedentedly low detection limit of 1.6 × 10−21g/L. Through monitoring the fluorescence quenching of AFM1with Fe3+addition, the interaction between them has been identified at a ratio of 1:936. Transient fluorescence analysis reveals that the fluorescence quenching process is predominantly static. It is interesting that the application of iron chelator diethylenetriaminepentaacetic acid (DTPA) cannot prevent the interaction between AFM1and Fe3+. With a particle size distribution analysis, it is suggested that a combination of AFM1and Fe3+occurs and forms a polymer-like aggregate. Nonetheless, the mutual reaction mechanism between AFM1and Fe3+remains unexplained and urgently necessitates unveiling. Finally, the developed sensor is successfully applied for the AFM1test in real samples, fully meeting the detection requirements for milk.