Abstract: This paper reported the preparation of antibody of Fusarium toxin Deoxynivalenol (DON) and establishment of indirect ELISA for DON. The optimal concentration of mice serum dilutions and the coated antigen required to observe an absorbance of 1.0 in the indirect competitive ELISA were 1/1600 and 1/1500 respectively. Color development in the assay was inhibited 50% by 63μg DON/ml, 114μg 3-Ac-DON/mL and more than 1000μg T-2/mL respectively. The Cross-reactivity to DON, 3-Ac-DON and T-2 of the antiserum were 100%, 55.2% and less than 6.3%. The effect of methanol concentration on the reaction between the antiserum and DON was studied. The result showed that the assay could be performed satisfactorily using an extraction solvent consisting of less than 10% methanol. Meanwhile, indirect competitive ELISA for detection of DON in wheat seeds was developed with the antibody. The range for detection of DON was 0.1-100μg/ml and the minimum detected concentration was 0.1μg/mL. Recoveries averaged 82%-93% with a coefficient of variation of 4.65%-21.3%. Analysis of 4 samples for DON exhibited positive results.