The invention discloses a preparation method of anti-fluoroquinolone rabbit monoclonal antibodies and application thereof. Six fluoroquinolone medicaments are chosen in the invention and coupled with Bovine serum albumin (BSA) to synthesize immunoantigens respectively. The above six immunoantigens are injected subcutaneously in the backs of immune rabbits after being mixed together. The anti-fluoroquinolone rabbit monoclonal antibodies are obtained by hybridoma technique. Rabbit monoclonal antibodies, which are superior to rat monoclonal antibodies, are prepared in the invention. More antigenic determinants are identified when rabbits are chosen as the experimental animal instead of rats. In addition, more integrated experiments can be carried out in rabbit spleens, since the spleens of rabbits are larger than that of rats. The invention provides the possibility of high throughput screening for fusion cells. The anti-fluoroquinolone antibodies of wide-adaptability in the invention has good detection effect on fluoroquinolones, such as enrofloxacin, ofloxacin, Difloxacin, Pefloxacin, Fleroxacin, Danofloxacin, etc., thus making it applicable to fluoroquinolone residue detection on food, forage and environmental samples.