Abstract: It was developed a chemiluminescence immunoassay (CLIA) method based on polyclonal antibody to detect the enrofloxacin residue in animal food. The CLEIA conditions were optimized. The optimal concentration of coating antibody dilution was 1∶64000 and the enrofloxacin markered enzymes dilution was 1∶256000. The results showed that:with an concentration range from 3 to 810pg/mL, the regression equation was y=-13.898x+ 110.38 (R2=0.9893) . The 50% inhibition percentage (IC50) and sensitivity (IC10) was 69.63pg/mL and 1.41pg/mL, respectively. Actual sample testing experiment results showed:in chicken, fish, shrimp, honey and milk samples, the detection limits was 3.76, 4.59, 2.85, 2.65 and 4.20pg/mL, respectively. The limit of quantity was 6.13, 7.35, 3.57, 3.73 and 6.48pg/mL, respectively. Recoveries were between 88.28% ¹02.6% when ENR was spiked to samples at 50¹000pg/g (pg/mL) . The mean intra-assay variability and inter-assay was 2.61% and 4.71%, respectively. The CLIA which we stablished could be used to detect enrofloxacin residues in animal food.