Abstract: The enzyme-linked immunosorbent assay (ELISA) for the detection of diethylstilbestrol (DES) was established, the most suitable working conditions of ELISA were optimized. The optimal concentration of the coating antigen (DES-OVA) and optimal dilution of monoclonal antibody against DES were 2 μg/mL and 1∶3×104，respectively. The linear regression equation of the standard curve was R2=0.9956. The range of linearity was from 0.125 to 128 ng/mL. The 50% inhibition value (IC50) was 1.103 ng/mL, sensitivity of the method was 0.077 ng/g，respectively. The intra-assay and inter-assay CVs were 6.14% and 7.48%. The recoveries of mud eel ranged from 77.6% to 94.6%. The very high specificity was evaluated by the significant cross-reactivity with analogous compounds and other aquatic antibiotics，the McAb against DES had 112.7% cross-reactivity with diethylstilbestrol-mono-carboxylic propyl ether，there was no cross-reactivity with other aquatic antibiotics(＜0.1).

Key words: diethylstilbestrol; drugs residue; ELISA