Abstract: Aeromonas hydrophila and Vibrio fluvialis are the causative agents pathogenic bacterial of haemorrhagic septicemia of a variety of farmed fish in China. Abivalent vaccine against these two pathogens was being developed by an orthogonal array design. The optimized bivalent vaccine could elicit effective protection from experimental challenge of A. hydrophila for C. auratus 4 weeks after immunized by i.p injection, but not by neither immersion vaccination nor hyperosmotic infiltration with or without supplement of polysaccharide. The vaccine was also protective against the challenge of V. fluvialis when fish was immunized by i.p injection and immersion vaccination plus polysaccharide. At challenge time, serum agglutination titers from fish injected with the bivalent vaccine were 1∶4-1∶32, 1∶2-1∶16, results of immunodiffusion indicated that there were two dominant antigens in boiled and sonicated V. fluvialis and A. hydrophila vaccine.