Pseudomonas stutzeri strain XL-2 exhibited significant performance on biofilm formation. Extracellular polymeric substances (EPS) secreted by strain XL-2 were characterized by colorimetry and Fourier transform infrared (FT-IR) spectroscopy. The biofilm growth showed a strong positive correlation (r(P)=0.96, P<0.01) to extracellular protein content, but no correlation to exopolysaccharide content. Hydrolyzing the biofilm with proteinase K caused a significant decrease in biofilm growth (t=3.7, P<0.05), whereas the changes in biofilm growth were not significant when the biofilm was hydrolyzed by alpha-amylase and beta-amylase, implying that proteins rather than polysaccharides played the dominant role in biofilm formation. More specifically, confocal laser scanning microscopy (CLSM) revealed that the extracellular proteins were tightly bound to the cells, resulting in the cells with EPS presenting more biofilm promotion protein secondary structures, such as three-turn helices, beta-sheet, and alpha-helices, than cells without EPS. Both bio-assays and quantitative analysis demonstrated that strain XL-2 produced signal molecules of N-acylhomoserine lactones (AHLs) during biofilm formation process. The concentrations of C-6-HLS and C-6-oxo-HLS were both significantly positively correlated with protein contents (P<0.05). Dosing exogenous C-6-HLS and C-6-oxo-HLS also resulted in the increase in protein content. Therefore, it was speculated that C-6-HLS and C-6-oxo-HLS released by strain XL-2 could up-regulate the secretion of proteins in EPS, and thus promote the formation of biofilm.