A detection method for detecting HPLC-ESI-MS/MS of 19 species of quinolone drugs simultaneously belongs to the antibiotic drug analysis technology field. The detection method comprises three steps: sample treatment, liquid-phase separation and mass spectrometric detection, wherein the 19 species of quinolone antibiotics in a tissue fluid is subjected to positive ion MRM scan; norfloxacin-D5 is taken as the internal standard; C18 chromatographic columns are adopted for separation; water-methanol containing formic acid is taken as the mobile phase for gradient elution; and the eluent is subjected to mass spectrometric detection. The detection method is characterized in that the organic solvent extraction method is adopted for carrying out the sample pretreatment according to the practical situation of the sample and the different detection sensitivity requirements; norfloxacin-D5 is taken as the internal standard; the C18 chromatographic columns are adopted for separation; and the water-methanol containing formic acid is taken as the mobile phase for gradient elution. The pretreatment process of the method is rapid and convenient, the detection result is reliable and sensitive, and the method is suitable for the analysis and the detection of the quinolone drug residues in animal food.