Abstract: Difloxacin (DIF) was coupled with carrier protein bovine serum albumin(BSA) and ovalbumin (OVA) to prepare complete antigen DIF-BSA and DIF-OVA by modified method of carbodiimide. Both UV scanning spectrum and SDS-PAGE were used to identify DIF artificial antigen. BALB/C mice were immunized with BSA-DIF, the titre of monoclonal antibody (mAb) was derected by indirect ELISA and blocking ELISA. The hybridoma lines that secrete DIF mAb were established with using hybridoma technology. The immunological traits such as titer, affinity and specificity of the mAb were characterized.The results showed that the complete antigens DIF-BSA was successfully coupled and its the conjugantion ratio of DIF to BSA was about 4.7:1.There hybridoma cell lines of 1H10、2F12 and 4D8 were screened for specificity to DIF, the indirect ELISA titer of the mAb were 1:5.12×102、1：3.2×101、1：2.56×102 in supernatant, 1：1.6×105、1：4×104、1：8×104 in ascites, the isotypes of the mAb were IgG1、IgG1、IgG2a，and the affinity constant(Ka) were 2.94×1010 L/moL、1.72×1010 L/moL and1.35×1010 L/moL, respectively, the mAb of 1H10 showed good sensitivity with IC50 of 1.64ng/ml to DIF.mAb has no cress-reactivity to other FQS and sulfonamides. DIF mAb of high-titer, sensitivity and specificity had been generated, it is possible to establish immunoassay of DIF residues in animal food. Key words: Difloxacin; Artificial immunity; Monoclonal antibody; Immunological trait.