Abstract: Objective:A great accurate and repeatable method for the determination of tetracycline(TC)by competitive chemiluminescence enzyme immunoassay assay(CLEIA)was established. Method:The TC-CLEIA,which was used to detect the concentration of TC,was established by choosing appropriate coupling carrier,optimizing reaction temperature and reaction time. Results:The detection range of TC-CLEIA for detection TC was 0.0259~95.9478 μg/L with 0.9998 of r. And the detective limit was 0.0133 μg/L,accuracy was 99.17%,sensitivity was 0.7305 μg/L and variation coefficient was less than 10%. There was little cross-reaction with chloramphenicol and penicillin in TC-CLEIA. The detection results had higher accuracy and repeatability when the different concentration of TC standard was added into the blank pork,fish and liver of chicken. The concentrations of TC residues in liver,kidney and muscle detected by TC-CLEIA using tetracycline-toxic mice model. The results showed that the order of concentration was kidney > liver > muscle. Conclusion:The TC-CLEIA method with high sensitivity and specificity was suitable for the detection of TC residues in animal-derived foods.