Abstract: Total RNA was separated from the abdomen of house fly,after which had been challenged for 24 hours with E.coli and Staphylococcus aureus.Then,the mR NA was purified from it.After that,ds-cDNA was synthesized by reverse transcriptase from mRNA,and small cDNA which was lower than 400 bp was removed by Sep harose CL-4B spun column.After EocRⅠ/NotⅠAdaptors were added to cDNA,those that werent ligated to cDNA were removed by another Sepharose CL-4B spun column.ThecDNA was inserted into λgtll,the recombined vector packaged in vitro,infected a host strain Y1090.Thus,the cDNA library was co structed.The titer of the newly constructed cDNA library was 3.46×105 pfu/mL,and its recombination rate was 99.6%.The library would provide basis for t he cloning of the antimicrobial peptide genes of house fly.