Hemolysin is a significant toxin secreted by Aeromonas hydrophila, which contributes pathogenicity of fish to humans. The complete ORF of hemolysin gene (1886 bp) was amplified using PCR. It was cloned in TA and sub-cloned in pET28a vector then transformed into Escherichia coli BL21(DE3) codon plus RP cells expressed by the induction with 1.0 mM of IPTG. The expected size of expressed protein was 68.0 kDa estimated by migration in 12% SDS-PAGE. Anti-His monoclonal antibodies were used to substantiate the recombinant protein by Western blotting. The percent similarity between hemolysin of A. hydrophila with other hemolytic toxins revealed that the hemolysin/aerolysin/cytotoxin sequence varied from 99.35 to 50.40%. Homology modeling was used to construct 3-D structure of hemolysin of A. hydrophila with the known crystal 3-D structure (PDB: 1XEZ). This protein can be used for immunoassays and it is suitable for vaccine candidate against A. hydrophila infection.