Abstract A fast and simple electrochemical method for the determination of diethylstilbestrol (DES) was developed. Gold nanoparticles stabilized by chitosan (AuCS) were prepared and used in combination with L-cysteine (L-Cys) by self-assembly for fabricating the electrochemical sensors. Compared with unmodified electrode, the oxidation peak current of DES could be increased greatly by using the L-Cys/AuCS modified GCE (L-Cys/AuCS/GCE), indicating L-Cys/AuCS had excellent ability to promote electron transfer. Under the optimal conditions, the oxidation peak current was linear to the concentration of DES in the range from 5 × 10−7 to 1 × 10−4 mol/L (r = 0.997) with the detection limit of 1.7 × 10−7 mol/L (S/N = 3). The proposed method was successfully used for the determination of DES in livestock and poultry meats with the satisfying recovery results (75.3–91.9%). The method not only exhibited good accuracy and reproducibility for DES detection, but also had the advantages of low cost, simple preparation method and convenient for batch preparation.