Abstract: The purpose of this experiment was preparing kanamycin completely artificial antigens. Firstly, kanamycin standard was reacted with bovine serum albumin (BSA) or ovalbumin (OVA) to prepare kanamycin artificial antigens. Two different coupling methods carbodiimide (EDC) and glutaraldehyde (GDA) were respectively prepared KAN-BSA (as an immunogen) and KAN-GDA-OVA (as the coating antigen). Both of the SDS-PAGE gel electrophoresis and the animal immunization experiment identified the antigen-coupled case. The results showed that, the prepared kanamycin complete antigen could stimulate the bodies to produce antibodies in mice, and the antiserum titer reached 1:2.56×10⁴, The IC₅₀ of the mice was 22.28 ng/mL. The results illustrated the immunogenicity of the complete antigens were good and the preparations of the antigens were successful.