This paper presents the generation of monoclonal antibodies (mAbs) with high specificity against marbofloxacin (MAR) through cell fusion procedures, and the development of a mAb-based indirect competitive ELISA (icELISA) method to detect MAR residue using one of these Hybridomas (clone M3C2). Under the optimal experimental conditions, this assay exhibited a working range of 0.008–48 ng/mL with IC 50 and LOD values of 0.16 and 0.005 ng/mL, respectively. Except for a slight cross-reactivity (12.8%) to ofloxacin, negligible cross-reactivity to the other compounds was observed. Recovery studies indicate an excellent correlation between concentration spiked and concentration determined in chicken. Therefore, the selected hybridomas put a potential for the development of a rapid test kit and provide an alternative method for the detection of MAR residues in food producing animals.