The invention discloses a fluoroquinolones-resistant specific monoclonal antibody hybridoma cell strain, a monoclonal antibody generated based on the cell strain, and the application thereof. The preservation number of the cell strain is CCTCC No.C2015221. The cell strain can stably secrete a monoclonal antibody specific to fluoroquinolones. The result of an indirect ELISA test shows that, the monoclonal antibody is high in sensitivity and strong in universality for the drug detection of ciprofloxacin, enrofloxacin, norfloxacin, pefloxacin, enoxacin and the like. The monoclonal antibody is applied to other immunoassay products, such as the preparation of immunogold labeling cards, immune affinity chromatography gels and the like. Obtained products are still high in sensitivity, high in selectivity, strong in enrichment and strong in universality for target detection. Therefore, the monoclonal antibody can be widely applied to the trace residual qualitative and semi-quantitative analysis of mainly fluoroquinolones in the fields of agriculture, food, environment and the like.