Abstract: This article aimed to establish an amplified luminescent proximity homogeneous assay linked immune sorbent assay (AlphaLISA) for detecting quinolones (QNS) residues in milk. The EDC/NHS active ester method was used to connect 6-aminocaproic acid as the spacer arm on the norfloxacin molecule to prepare biotin-modified norfloxacin hapten (Biotin-6AS-NOR), and QNS monoclonal antibody was coupled on the receptor microsphere. Biotin-6AS-NOR could compete with QNS in the sample to bind quinolone antibodies. A competitive indirect AlphaLISA for QNS residues in milk was established by optimizing the parameters including QNS antibody coupling amount on receptor microspheres, Biotin-6AS-NOR, the dosage of donor/receptor microspheres, and dilution schemes for milk samples. By using the 1:5 milk sample dilution scheme, the limit of detection and the limit of quantification of the established AlphaLISA for QNS in milk were 0.19 and 0.46 ng/mL, respectively. The recovery values ranged from 85.97% and 110.11%, and the intraday and interday variation were both less than 10%. The cross-reactivity values for norfloxacin, enrofloxacin, ciprofloxacin, ofloxacin, pefloxacin, ofloxacin, and lomefloxacin were all higher than 70%. In conclusion, the established AlphaLISA showed high sensitivity, accuracy and good repeatability, without the need of tedious wash steps throughout the detection process; therefore, it is easy to achieve automated detection of chemical hazards residue in milk and has high application and promotion value.