Biofilms are a form of microbial community that can be beneficial for industrial fermentation because of their remarkable environmental resistance. However, the mechanism of biofilm formation in Saccharomyces cerevisiae remains to be fully explored, and this may enable improved industrial applications for this organism. Although quorum-sensing (QS) molecules are known to be involved in bacteria biofilm formation, few studies have been undertaken with these in fungi. 2-phenylethanol (2-PE) is considered a QS molecule in S. cerevisiae. Here, we found that exogenous 2-PE could stimulate biofilm formation at low cell concentrations. ARO8p and ARO9p are responsible for the synthesis of 2-PE and were crucial to the formation of biofilm. Deletion of the ARO8 and ARO9 genes reduced the content of 2-PE in the early stage of fermentation, reduced ethanol yield and decreased biofilm formation. The expression of FLOp, which is involved in cell adhesion, and the content of extracellular polysaccharides of mutant strains Delta ARO8 and Delta ARO9 were also significantly reduced. These findings indicate that the production of 2-PE had a positive effect on biofilm formation in S. cerevisiae, thereby providing further key details for studying the formation of biofilm mechanism in the future.