OBJECTIVE It is reported that Streptococcus mutans luxS gene may have an important role in the interspecies quorum sensing system. To construct the S. mutans luxS gene knockout mutant, this research aim to construct the luxS gene allelic exchange plasmid. METHODS The upstream and downstream flank DNA fragments of S. mutans luxS gene (Xup, Xdn)and the E. coli kanamycin resistance gene (Kana) were enriched by pfu DNA polymerase with "nest PCR" methods. These fragments were ligated into pBluescript SK (+) Phagemids vector with double endonuclease reaction sequentially. RESULTS With endonuclease reaction and DNA sequencing, it was proved that the objective plasmid, Xukd-pbsk, was constructed correctively and the kanamycin resistance gene could be expressed in vitro. CONCLUSION The S. mutans luxS gene allelic exchange plasmid is constructed correctively in this research and can be used in the future research of S. mutans luxS gene knockout mutant.