Abstract: The aim of the study is to establish a method for determination of amantadine residues by chemiluminescent enzyme immunoassay (CLEIA). After optimizing the dilution multiple of enzyme-labeled antigen and magnetic labeled antibody, reaction time and substrate luminescence time, a standard curve was established, and finally the detection limit, accuracy and precision of the method were evaluated. The results showed that the 50% inhibitory concentration (IC₅₀) was 0.481 μg/L, and the linearity range was 0.085~2.729 μg/L. The detection limit in animal tissues, veterinary drugs and feed were 0.1 μg/kg. The recoveries ranged from 80.2%~94.5%, with the intra-assay and inter-assay coefficients of variation less than 10%. The method is sensitive, simple and it can be widely used for the determination of amantadine residues in animal tissues, veterinary drugs and feed.