This paper presents the generation of monoclonal antibodies (mAbs) with high specificity against enrofloxacin (ENR) through cell fusion procedures, and the development of a mAb-based indirect competitive ELISA (icELISA) method to detect ENR residue using one of these Hybridomas (clone 4B5-D6). Under the optimal experimental conditions, this assay exhibited a working range of 0.004-38 ng/mL with IC50 and LOD values of 0.4 and 0.002 ng/mL, respectively. Except for a high cross-reactivity (105.2%) to Ciprofloxacin, negligible cross-reactivity to the other compounds was observed. After optimization, 10% of methanol was used in the assay buffer and this ELISA system can tolerate acetonitrile not higher than 10%. Recovery studies indicate that an excellent correlation between concentration spiked and concentration determined was found, and the results also suggest this assay has the potential to be incorporated into a quantitative monitoring program for the rapid screening of ENR residue in poultry tissues.