Abstract: Objective To construct plasmid of fibrinogen-binding proteins gene with fnbA，fnbB and fnbAB knockout and then to construct corresponding gene deletion staphylococcus aureus strains . Methods We first extract ed genome of staphylococcus aureus strain NCTC8325. Then we designed and synthesized the specific primers containing GGGG -attB1 gene sequence. Purpose gene?fragments were made?G ateway?compatible with adapter primers and amplified by PCR. These fragments were used in a BP clonase reaction to create entry clones. The created pKOR1 plasmid vector containing knockout fnbA, fnbB and fnbAB were transferred into staphylococcus aureus strains NCTC8325 with electroporation. Results The sequencing results of plasmid pKOR1fnbA, pKOR1fnbB and pKOR1fnbAB were correct . It has also been verified by PCR that plasmid pKOR1fnbA, pKOR1fnbB and pKOR1fnbAB were all successfully transfered into staphylococcus aureus strain s NCTC8325 with electroporation. Conclusion We successfully constructed staphylococcus aureus strains NCTC8325 with fnbA, fnbB knockout and 2 genes double knockout, which provide powerful tools for studying the role of fnbA and fnbB gene in the mechanism of staphylococcus aureus infection .