The biofilm formation of Lactobacilli is regulated by the LuxS/AI-2 quorum sensing (QS) system, but the mechanism of QS regulating the formation of Lactobacilli biofilm is not clear. This study aimed to investigate the mechanism of producing biofilm in L. plantarum R and its effect on the quality of fermented pickles based on LuxS/AI-2 QS system. Compared with L. plantarum R, the AI-2 activity of L. plantarum RΔluxS was significantly reduced, but the biofilm, extracellular protein, and eDNA were significantly increased. Moreover, expression of oppA, livJ, livH and comD genes was up-regulated and luxS, peg.3090 and peg.3093 was down-regulated. Results showed that peg.3093 was most significantly down-regulated in L. plantarum RΔluxS, and extremely significant negatively correlated with biofilm. The biofilm, eDNA, and extracellular protein of L. plantarum RΔpeg.3093 was higher than those of L. plantarum R. Moreover, metabolomics showed that deletion of luxS gene could decrease AI-2 level, promote anthocyanin and flavonol biosynthesis, lead to improving the antioxidant properties and quality of pickles. Thus, luxS gene knockout may increase biofilm by down-regulating the expression of peg.3093 to increase extracellular protein and eDNA. This study provides a theoretical basis for the enhancement of Lactobacillus biofilm and its application.