The focus of this study was to investigate the effects of luxS, a key regulatory gene of the autoinducer-2 (AI-2) quorum sensing (QS) system, on the biofilm formation and biocontrol efficacy against Ralstonia solanacearum by Paenibacillus polymyxa HY96-2. luxS mutants were constructed and assayed for biofilm formation of the wild-type (WT) strain and luxS mutants of P. polymyxa HY96-2 in vitro and in vivo. The results showed that luxS positively regulated the biofilm formation of HY96-2. Greenhouse experiments of tomato bacterial wilt found that from the early stage to late stage postinoculation, the biocontrol efficacy of the luxS deletion strain was the lowest with 50.70 +/- 1.39% in the late stage. However, the luxS overexpression strain had the highest biocontrol efficacy with 75.66 +/- 1.94% in the late stage. The complementation of luxS could restore the biocontrol efficacy of the luxS deletion strain with 69.84 +/- 1.09% in the late stage, which was higher than that of the WT strain with 65.94 +/- 2.73%. Therefore, we deduced that luxS could promote the biofilm formation of P. polymyxa HY96-2 and further promoted its biocontrol efficacy against R. solanacearum.