Abstract: The Ciprofloxacin (Ciprofloxacin, CIP) was conjugated to carrier protein BSA and OVA by EDC to obtain the artificial and coating antigen . The ultraviolet scanning and SDS-PAGE were used to identify Ciprofloxacin artificial antigen. BALB/c mice were immunized with BSA-CIP. The titer of polyclonal antibody (pAb) was detected by indirect ELISA and blocking ELISA after three times immunization. The high titer, sensitive and specific mouse was selected for cell fusing. The hybridoma lines that secrete CIP mAb were established with using monoclonal antibody hybridoma technology and the immunological characteristics such as titer, sensitivity and specificity of the mAb were characterized. SDS-PAGE results showed that CIP artificial antigen was synthesized successfully. Three BALB/c mice indirect ELISA titer against CIP were all above 10-4 and the IC50 of No. 2 mice was 48.59μg/L. Two hybridoma cell lines of Z3G12B3 and Z2H8C10 were screened for specificity to CIP after cell fusing, the indirect ELISA titer of the mAb were 1:512 in supernatant, 1:2.56×105 in ascites, the mAb of Z3G12B3 showed good sensitivity with an IC50 of 2.47μg/L to CIP, had less than 0.03％cross-reactivity with sarafloxacin and difloxacin, and no cross-reactivity to other compounds. The high-titer, sensitive and specific CIP mAb has been generated and laid a solid foundation for the CIP ELISA kit and strip.