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A simple and effective direct competitive chemiluminescence immunoassay for detection of four kind of quinolone antibiotics in milk was established using Nor-Biotin(biotin-modified Norfloxacin) bifunctional ligand and SA-ALP signal amplification technology. The polyclonal antibody was obtained after immunization of New Zealand White rabbits using Norfloxacin-derived antigen. ”Click chemistry” was used for the rapid and facile synthesis of the Nor-Biotin bifunctional ligand. After the optimization of the incubation time and reaction buffer, the direct competitive ChemiLuminescence assay (dcCLIA) method was developed and used for sensitive detection of four kinds of quinolone drugs (norfloxacin (NOR), pefloxacin (PFLX), ciprofloxacin (CIP), and danofloxacin (DFLX)). The IC50 of the four kinds of quinolone drugs ranged from 7.35 ng/mL to 24.27 ng/mL, and the lowest detection limits (LOD) ranged from 0.05 ng/mL to 0.16 ng/mL, which were below their maximum residue levels (MRLs), approved by the EU for treatment of food-producing animals. To demonstrate the applicability of the assay, artificially contaminated milk samples with the four-quinolone drugs were analyzed. The mean recovery rates of the drugs ranged from 86.31% to 112.11%.