Abstract： Objective To carry out gene cloning, sequence analysis and recombinant expression vector construction of CMY-type AmpC β-lactamase from two strains of Citrobacterfreundii. Methods Total genomic DNA from Citrobacterfreundii strain 30, 31 which produced CMY-type AmpC β-1aetamase acted as templar, the blaCMY was amplified by PCR. Nucleotide sequence measure was performed after blaCMY had being cloned in pGEM-T vector. Plasmid conjugation test were examined between strain 30, 31 and E.coli HBIO1Rif. And then the blaCMY was cloned into pBV220 vector. AmpC induce tests of strain 30, 31 and recombinant strain were detected. Results 1146 bp DNA fragment of CMY-type AmpC β-lactamase by PCR showed 97% amino acid identity with blaCMY which already registered in GenBank. Plasmid conjugation test proved blaCMY located in plasmid and plasmid could conjugate. The results of three-dimension test showed AmpC β-1actamase from strain 30, 31 and recombinant strain could hydrolyze cefoxitin. Conclusion The blaCMY from strain 30, 31 was a novel gene subtype of CMY- type AmpC β- laetamase. Recombinant expression vector pBV220/CMY is successfully constructed, which provides good evidence for further study of expression and purification of blaCMY.