To construct LuxS deletion mutant of Streptococcus mutans, and study the effect of luxS mutation on the biofilm formation of streptococcus mutans under various conditions, and find out the differences between luxS mutant strain and streptococcus mutans. Long flanking homology polymerase chain reaction(LFH-PCR) was introduced to generate a gene disruption construct consisting of Emr cassette with long flanking homology regions to the target gene. The electroporation competence of Streptococcus mutans was then transformed with this PCR product. Then positive transformants were counted on selective agar which containing erythromycin and identified by PCR. The streptococcus mutans-luxS mutant and the standard strain were grown in three different conditions(BHI, 2% glucose-BHI, 2% saccharobiose-BHI), and the ability of S.mutans and LuxS mutant biofilm formation was examined in 24 h by scanning electron microscopy (SEM). Identification by PCR and sequencing confirmed the validity of the LuxS deletion mutant of Streptococcus mutans. Compared with S.mutans ,the LuxS mutant maintained with 2% sucrose displayed an apparent defect in biofilm formation. Conclusions: The successful construction of the LuxS deletion mutant, and the ability of sucrose-dependent biofilm formation will be down-regulated in Streptococcus mutans after LuxS gene was knocked out.