Objective To prepare neutralizing monoclonal antibody of tetrodotoxin (TTX) and establishment of a detection method of the antibody based on competitive ELISA. MethodsBlab/c mice were immunized with TTX-KLH and its monoclonal antibody was detected by TTX-BSA coated indirect ELISA. A hybrydoma cell line was established and its ascites was injected into peritoneal cavity of the mice. The monoclonal antibody was purified with protein A sepharose CL4B affinity column and identified with SDS-PAGE electrophoresis and indirect ELISA. LD50 of tetrodotoxin for Kunming mice was determined by routine method. A mixture of monoclonal antibody and TTX was injected into the peritoneal cavity of mice, and the neutralizing capacity of monoclonal antibody was detected. A detection method of TTX was also established by competitive ELISA. ResultsTwo kinds of neutralizing monoclonal antibodies were obtained, and their purity was above 95% obtained by Protein A sepharose CL 4B affinity column. Monoclonal antibody 5E7 had a higher binding capacity with TTX than that of monoclonal antibody 5E4. The neutralizing antibody showed a protection rate of 50% for Kunming mice against 2 LD50 TTX attack. A detection method of TTX with the monoclonal antibody based on competitive ELISA was established and the detectable minimal concentration of TTX was 1.56 μg/mL. Conclusion Two kinds of neutralizing monoclonal antibody have been obtained. The protection rate was 50% for Kunming mice against 2 LD50 TTX attack. A detection method of neutralizing monoclonal antibody established based on competitive ELISA, with a minimum detection limit of 1.56 μg/mL.