In order to further develop the method for detecting residual Clenbuterol（CL），so as to lay foundation for research and development of the detection kits，monoclonal antibodies（mAbs）against CL were prepared. Through diazo reaction，CL was coupled with Bovine serum albumin（BSA）and Ovalbumin（OVA），the coupling products were used as immunogen and detection antigen，respectively. Using hybridoma technique，5 monoclonal hybridoma cell strains（1E9A9、2A9C1、3F2E9、6G2E9、6E10D9）which could secret antibodies against small CL molecules were obtained. During serial subcultivation in vitro and cryopreservation and resuscitation，the antibodies could be secreted stably. ELISA detection showed the antibody titers of supernatant of the 5 strains were 1:128 000，1:512 000，1:128 000，1:512 000 and 1:256 000，respectively. Ascites containing mAbs were induced by hybridoma cell strain 3F2E9 and 6E10D9 and the antibody titers in purified ascites were 1:128 000 and 1:256 000. Measurement by BCA protein determination method showed their concentrations were 1.60 mg/mL and 1.54 mg/mL repectively. Through Dot-ELISA，good reactivity between the two mAbs and CL was verified. As a conclusion，specific mAbs against CL were prepared successfully，which would lay foundation for further research of the kits for CL residue detection.