In order to grope for feasibility and basic theory of preparing fusants bacterins of fish pathogeny, strains, Aerornonas punctta f.intestinalis 58-20-9 and Myxococcs piscicola G4 were employed as arch-strains to conduct protoplast fusion and establish fusants．When the temperature of enzymolysis was 37 ℃, the best enzymolysis time was singled out to be 40 minutes and the best lysozyme density to be 4 mg/mL．Under the condition of operating PEG as agent of accelerating fusion,Aeromonas punctta f.intestinalis 58-20-9 strains and Myxococcs piscicola G4 strains performed a protoplast fusion, and their protoplast formation rates were 54.7%, 50.8% respectively and their protoplast regenerative rates of 58-20-9 strains were 18.2% and 12.2% respectively. Arch-screening protoplast fusion rates were 4‰．After dibbling seeds of 500 fusants colony from arch-screening on duplex repelling selected culture medium and generating continuously 12 times, a steady fusant colony could be derived, whose steady rates were 0.2%.