Abstract: Edwardsiella piscicida, belonging to the family Enterobacteriaceae, is a Gram-negative intracellular pathogen. It causes hemorrhagic septicemia in more than 20 important species of farmed fish, especially flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus), leading to huge economic losses. In dissecting the host-pathogen interaction, antibodies against virulent proteins of E. piscicida are often required. It always takes a long time to prepare antibodies in mice or rabbits, and the polyclonal or polypeptide antibodies obtained are often not specific, especially when applied in eukaryotic cells. To resolve this problem, the λ Red recombination method established in E. coli and Salmonella was optimized to introduce epitope tagging to chromosomal genes in E. piscicida, by using linear DNA fragments for homologous recombination with genomic DNA. Plasmid (pKD3/pKD4, pSU312/pSU313, pSU314/pSU315, or pSUB7) was used as the template, primers are composed of gene specific sequence plus FRT sequence. The DNA fragment obtained by PCR was introduced into E. piscicida transformed with pKD46. With the supplementation of L-arabinose, pKD46 expresses three recombinant proteins (Exo, Beta, Gam) of λ phage, under which the DNA fragment introduced recombine homologically with E. piscicida genome, resulting in target gene deletion or a commercial tag insertion. To eliminate the antibiotic gene introduced in this process, pKD46 expressing Flp recombinase (pKD46-flp) was constructed. By homologous recombination, the antibiotic gene between two FRT sites and one FRT site are eliminated. By culturing at 37℃, the pKD46-flp introduced is cured, obtaining an epitope tagging of chromosomal gene of E. piscicida. A commercial monoclonal antibody against the tag (HA, FLAG, or His) could then be used to monitor the expression or translocation of virulent proteins from E. piscicida. The method established in this study facilitates the dissection on host-pathogen interaction, and will shed light on the genetic operation on other aquatic bacterial pathogens.