Abstract: The artificial ciprofloxacin-bovine serum albumin (CPFX-BSA) was inactivated as antigen to immunizate Balb/c mice. with hybridoma technology, One strain hybridomas secreting McAb against CPFX-BSA named CPFX 5G4 was obtained ultimately. The results showed that the titer of CPFX 5G4 was 1.28×10-6; the molecules of the Heavy chain and Light chain of the McAb were approximately 50 KD and 25 KD according to SDS-PAGE electrophoresis and the antibodies subtypes was IgG3. A ic-ELISA calibration curve was drawn up according to established system. It was calculated that the IC50 value was 192.44 ng/mL and the detection limit was 16.29 ng/mL. The rate of cross reaction between McAb-CPFX and enrofloxacin was 62.45%. The McAb-CPFX laid a foundation for the study and assembly of the rapid test kit.