A rapid and sensitive method based on biotin-avidin mediated competitive enzyme-linked immu nosorbent assay(BA-ELISA) was established for the determination of ketamine.The optimal concentration of coated antigen and anti-ketamine monoclonal antibody were found to be 2.0 and 10.2 mg/L.The concentra tions of biotinylated goat anti-mouse IgG(Biotin-IgG) and streptavidin-horseradish peroxidase(SA-HRP) were optimized and the optimum results were found to be 0.29 and 1.0 mg/L, respectively.The linear range of the presented method was from 0.1 to 1000 μg/L, and the limit of detection was found to be 0.03 μg/L.The recoveries of ketamine spiked in human serum and urine were between 94% and 102%.Comparing the result of traditional ELISA, the present BA-ELISA method had a lower detection limit for ketamine.The experimental results indicated that the present BA-ELISA method was specific and sensitive.