A rapid indirect competitive ELISA (icELISA) format has been developed for the determination of enrofloxacin (ENR) residues in chicken. For this purpose, carbodiimide active ester method was employed to synthesize the artificial antigen of ENR-BSA, and anti- serum produced from the immunized rabbits was tested by indirect ELISA and icELISA. By the square matrix titration, the icELISA method was developed for the quantitative detection of ENR, based on the pAb. The Linear range was from 0.006 to 31.5 ng/mL, with LOD and IC50 value of 0.003 ng/mL and 0.45 ng/mL, respectively. Except for a high cross-reactivity (105.2%) to Ciprofloxacin, negligible cross-reactivity to the other compounds was observed. The recoveries of ENR were in the range of 98.3-127.5%, 85.7-112.5% and 97.4-103.8% for chicken muscle, liver and kidney, respectively. After optimization, 0.03 mol/L of HCl was used in the assay buffer and this ELISA system can tolerate acetonitrile not higher than 10%. The correlation coefficients (R2) of the ELISA and LC-MS data were 0.9472 in muscle, 0.9843 in liver, and 0.9382 in kidney. Therefore, this assay has the potential to be incorporated into a quantitative monitoring program for the rapid screening of ENR residue in food.