Aerolysin is a significant virulent toxin protein secreted by Aeromonas hydrophila; it produces deep wound infections and hemorrhagic septicemia. The complete aerolysin gene (1,482 bp) was amplified from A. hydrophila. Furthermore, it was cloned and expressed into Escherichia coli BL21(DE3) codon plus RP cells using 0.5 mM IPTG for induction. The protein size was 54 kDa as estimated by SDS-PAGE, and it was purified by Ni-NTA affinity chromatography. Anti-His antibodies were used to characterize the expressed aerolysin by Western blotting and showed hemolytic activity with fish red blood cells. Aerolysin may be used as immunoassays for earlier control of A. hydrophila and is also compatible for vaccination.