Abstract: The goal of this study was to develop a method for preparation of monoclonal antibody against Gonyautoxins GTX1-4. GTX1-4 artificial antigen was synthesized by formaldehyde method, and GTX1-4 monoclonal antibody was prepared by mouse immunity and monoclonal antibody technology. The results showed that the conjugated ratios of immunizing antigen GTX1-4-BSA and detected antigen GTX1-4-KLH were 10.7∶1 and 22.4∶1, respectively. Three hybridomas cell lines, 1-A4, 5-A11 and 5-E11, which could stably secret GTX1-4 monoclonal antibody were screened. The subtypes of monoclonal antibodies in ascites induced by 5-E11 and 1-A4 were IgG1 and IgG2a, and the titers of them were up to 64000. The subtype of monoclonal antibody in ascites induced by 5-A11 was IgG1, and its titer could be 128000. The sensitivity of monoclonal antibody in ascites induced by 5-A11 was 6.25 μg/mL, and this antibody has a strong specificity. In the study, consequently, GTX1-4 artificial antigen was successfully synthesized, and the hybridomas cell lines which could stably secret GTX1-4 monoclonal antibody was obtained.