A　simple　and　effective　direct　competitive　chemiluminescence　immunoassay　for　the　detection　of　4　kinds　of　quinolone　antibiotics　in　milk　was　established　using　Nor-Biotin　(biotin-modified　norfloxacin　[NOR])　bifunctional　ligand　and　alkaline　phosphatase-conjugated　streptavidin　signal　amplification　technology.　The　polyclonal　antibody　was　obtained　after　the　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　method　was　developed　and　used　for　sensitive　detection　of　4　kinds　of　quinolone　drugs　(NOR,　pefloxacin,　ciprofloxacin,　and　danofloxacin).　The　IC50　of　the　4　kinds　of　quinolone　drugs　ranged　from　7.35　to　24.27　ng/mL,　and　the　lowest　detection　limits　ranged　from　0.05　to　0.16　ng/mL,　which　were　below　their　maximum　residue　levels,　approved　by　the　EU　for　treatment　of　food-producing　animals.　To　demonstrate　the　applicability　of　the　assay,　artificially　contaminated　milk　samples　with　the　4　quinolone　drugs　were　analyzed.　The　mean　recovery　rates　of　the　drugs　ranged　from　86.31%　to　112.11%.　©　2021　The　Author(s)　2021.　Published　by　Oxford　University　Press　on　behalf　of　Japan　Society　for　Bioscience,　Biotechnology,　and　Agrochemistry.