Aims: The main aims of this study were to clone and express complete open reading frame (ORF) of thermostable direct haemolysin gene (tdh) from Vibrio alginolyticus strain HY9901 in Escherichia coli, and further evaluate the virulence of expressed TDH on mouse and crimson snapper. Methods and Results: A 410 bp internal fragment of the tdh gene was amplified by touchdown PCR with designed primers. Then its unknown flanking sequences of the 5'- and 3'-ends were finally characterized by inverse PCR and nested PCR. Sequence analysis showed that the tdh gene contain 570 bp ORF which encoded 189 amino acids. The deduced amino acid sequence of the ORF was in significant homology with several Vibrio TDH. The product that the tdh gene expressed in E. coli was purified by Ni2+ -IDA Sepharose affinity column. The activity of purified TDH was 4651 U mg(-1) protein by hide powder azure digestion. The lethal toxicity test showed that LD50 values of the purified TDH were 5.68 and 8.34 mu g TDH g(-1) body weight for mouse and crimson snapper, respectively. Conclusions: The complete ORF of tdh gene was obtained by touchdown PCR, inverse PCR and nested PCR. The ORF was perfectly expressed in E. coli. The activity and toxicity assays showed that the N-terminal signal pepticle was essential to autocatalyse and fold correctly to obtain the activity and toxicity in the purified TDH. The Native-PAGE analysis showed that the activated tdh gene expressed in E. coli was a dimer with two identical subunits. Significance and Impact of the Study: This study demonstrates that the expressed activated TDH can produce the toxicity protein determined on mouse and fish, which will lead to better understandings of the identifying virulence factor that could be considered as a candidate antigen for vaccine and a diagnostic tool for vibriosis. Its use as an immunizing antigen might prevent the ability of V. alginolyticus to infect the marine aquatic animals, as a complementary measure to tick control and appropriate management in countries affected by vibriosis.