The direct competitive enzyme-linked immunosorbent assay (dcELISA) kit was developed for detecting tetrodotoxin (TTX). The working conditions of the dcELISA kit including the anti-TTX mAb coating concentration, coating method, enzyme-labeled antigen concentration, the antigen diluents, reaction time and temperature were all optimized. The result showed that mAb coating concentration was 3.72 μg/ml, it was coated at the condition of minimal power treatment of microwave oven for 3 min. The enzyme-labeled antigen concentration was 4.08 μg/ml. The competitive reaction was under the condition of room temperature 25 °C for 30 min. The half maximal inhibitory concentration (IC50) of the standard curve was 20.4 ng/ml, detection limit was 1.1 ng/ml, linear range 3.3～137 ng/ml, the intra-assay CV and inter-assay CV were 6.25% and 7.34% respectively. And recovery rate of TTX ranged from 65.0% to 93.2% with the CV of 9.41～12.77%. This method is convenient, sensitive and time-saving, hope this dcELISA kit can bring benefits and reference for TTX detection.