Objective To explore the roles of type Ⅵ secretion system (T6SS) and quorum sensing (QS) system in the biofilm formation in Pseudomonas aeruginosa. Methods The PAO1 biofilm bacteria and planktonic bacteria were established. The expression levels of T6SS associated genes hemolysin co-regulated protein (Hcp) gene (Hcp1, Hcp2, Hcp3), QS associated gene LasR, exopolysaccharide associated genes polysaccharide synthesis locus A (PslA) and pellicle A (PelA), type Ⅳ pili gene pilus A (PilA) and flagellin C (FliC) were detected by quantitative real-time PCR, and the expression levels of those genes were compared between PAO1 biofilm bacteria and planktonic bacteria using independent t-test. Results The expression levels of PslA, PelA, PilA and FliC genes in PAO1 biofilm bacteria were respectively 714, 274, 604 and 42 folds of those in the planktonic bacteria (all P<0.05), suggesting that PAO1 bacteria formed mature biofilm with flagellum and pili. The expression levels of Hcp1, Hcp2, Hcp3, and LasR genes in PAO1 biofilm bacteria were respectively 1 045, 11 268, 6 654 and 1 226 folds of those in planktonic bacteria (all P<0.05), suggesting that T6SS and QS systems were related to the biofilm formation of PAO1 bacteria. Conclusion T6SS and QS systems might be involved in biofilm formation in Pseudomonas aeruginosa, and its regulatory mechanism needs further study.