Abstract: Serine protease (Spe) and hemolysin (Hly) are important pathogenic factors and protective antigens of Aeromonas hydrophila. The pathogen A. hydrophila TPS-30 strain was assigned to serogroup O: 9 of the Sakazaki and Shimada (National Institutes of Health) scheme, which caused hemorrhagic septicemia in Jiangsu Province. In this study, two pairs of primers were designed and synthesized according to the sequence of gene Spe and Hly. The Spe and Hly DNA fragments were amplified by PCR Spe from A. hydrophila TPS-30 genomic DNA respectively. The subsequent sequence analysis revealed the Spe gene of TPS-30 has an open reading frame of 1800 bp encoding a 600 amino-acid protein with a molecular weight of 66.6 kD, the Hly gene of TPS-30 has an open reading frame of 1410 bp encoding a 470 amino-acid protein with a molecular weight of 51.7 kD. Gene Spe and Hly were fused and cloned into expression vector pET32a (+) at the site of polyclone resulting to recombinant vector pET32a-Spe-Hly, gene Hly lying in 5' end and gene Spe in 3' end. The expression vector pET32a-Spe-Hly were transformed into Escherichia coli BL21 (DE3). After engineered, E. coli were incubated in LB medium at 37 ℃ for 18h, 1% inoculums volume were transferred into LB medium to incubate. When A600 reached 0.5, engineered E. coli were induced by the isopropyl-β-D-thiogalactopyranoside (IPTG) at ultimate concentration 0.2 mmol/L with 4h. Fusion protein (Hly-Spe) was expressed. The molecular weight of the fusion engineered protein was about 130 kD, which was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that Spe-Hly fusion protein expression vector pET32a-Spe-Hly was constructed successfully and the Spe-Hly fusion protein was highly expressed in recombinant strain BL21(DE3) (pET32a-Spe-Hly). To test the effectiveness of the recombinant Spe-Hly as a vaccine, Carassius auratus gibelio were immunised with the recombinant Spe-Hly and then challenged with different bacterial strains. The immune fish were significantly protected from challenges by the homologous strain (A. hydrophila TPS-30) and one serologically different strain (A. hydrophila BSK-10), giving RPS values of 81.4% and 73.1%, respectively, as compared with the control groups (injected with Montanide ISA763A and PBS). All these findings suggest that this recombinant Spe-Hly has the potential to be developed into an effective vaccine in fish against the infections of A. hydrophila.