A rapid and sensitive method based on chemiluminescent competitive indirect ELISA (CL-ciELISA) was developed for the screening of gentamicin (GEN) in milk. The cross-reactivity (CR) of gentamicin, sisomicin (SIS), neomycin (NEO), kanamycin (KAN), neamine (NEA), streptomycin (STR), apramycin (APR) and spectomycin (SPT) with this GEN-specific monoclonal antibody (MAb) produced in this experiment were 100, 3.2, <0.1, <0.1, <0.1, <0.1, <0.1, and <0.1%, respectively. In this method, IC50 (half maximal inhibitory concentration of a substance) of the MAb was 0.69 ng mL−1 and the limit of detection (LOD), measured by IC10, was 0.06 ng mL−1. This CL-ciELISA method with luminol solution as a substrate was 10 times more sensitive compared to the colorimetric-ciELISA. In spiked milk samples, the mean recoveries ranged from 85.6–97.9% with intra-day and inter-day variation less than 7.9 and 7.98%, respectively. The results illustrated that this procedure is applicable for screening gentamicin in milk.