Elastase LasB, an important extracellular virulence factor, is shown to play an important role in the pathogenicity of Pseudomonas aeruginosa during host infection. However, the role of LasB in the life cycle of P. aeruginosa is not completely understood. This report focuses on the impact of LasB on biofilm formation of P. aeruginosa PAO1. Here, we reported that the lasB deletion mutant (ΔlasB) displayed significantly decreased bacterial attachment, microcolony formation, and extracellular matrix linkage in biofilm associated with decreased biosynthesis of rhamnolipids compared with PAO1 and lasB complementary strain (ΔlasB(+)). Nevertheless, the ΔlasB developed restored biofilm formation with supplementation of exogenous rhamnolipids. Further gene expression analysis revealed that the mutant of lasB could result in the downregulation of rhamnolipid synthesis at the transcriptional level. Taken together, these results indicated that LasB could promote biofilm formation partly through the rhamnolipid-mediated regulation.