Abstract: Exonuclease I-based enzyme-linked aptamer assay for the detection of kanamycin residues in animal derived food was established in this paper. Aptamer binding with kanamycin could no longer be digested by exonuclease I, but could further specifically capture the horseradish peroxidase ( HRP) that catalyzed methyenedianiline substrate to produce color change. Determination of the limit of detection was based on linear relationship between absorbance value at 450 nm and the concentration of kanamycin. The effects of coated concentration, competitive reaction time, dosage of exonuclease I and blocking solutions on the detection were investigated. The results showed that under the optimal conditions, the established exonuclease I-depended enzyme-linked aptamer analysis of kanamycin have high sensitivity, the detection limit was 3.26μg/L with linear ranging from 5 to 100μg/L. When kanamycin of 5.0, 20.0 and 50.0μg/kg were added to four animal derived foods, the recovery rates ranged from 75.8% to 90.6%, the relative standard deviation for RSD ranged from4.2% to 7.8%. The method had simple operation without need large instrument and could be used for fast detection of kanamycin residues in animal derived foods.