To investigate the effects of cryptotanshinone on the growth and biofilm formation of Pseudomonas aeruginosa and its mechanism, the minimum inhibitory concentration (MIC) of cryptotanshinone on P. aeruginosa was determined by mi-crodilution, and the effect of cryptotanshinone on P. aeruginosa biofilm formation was observed by crystal violet staining, inverted microscope and laser scanning confocal microscope, and the effect of cryptotanshinone on P. aeruginosa biofilm for-mation was measured by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The effect of cryptotanshi-none on the expression of genes related to P. aeruginosa biofilm formation was detected by RT-qPCR. The MIC of cryptotan-shinone on P. aeruginosa was 800.0 μg/mL; after crystal violet staining, the best inhibition of biofilm was observed at the mass concentration of cryptotanshinone of 800.0 μg/mL in a dose-dependent manner (P<0.01). The formation and distribution of biofilm by cryptotanshinone were observed by confocal laser microscopy, and the inhibition effect was significantly enhanced at 200.0 μg/mL. In the range of 200.0～800.0 μg/mL mass concentration, cryptotanshinone at 800.0 μg/mL mass concentra-tion inhibited the expression of LasI, LasR, RhlI and RhlR genes (P<0.01). The mechanism of cryptotanshinone inhibition of P. aeruginosa biofilm formation may be through the down-regulation of LasI and other genes expression. The present study suggests that cryptotanshinone can be used as a group-sensing inhibitor to intervene in the drug resistance of P. aeruginosa, which can provide a reference for the treatment of multi-drug-resistant P. aeruginosa (MDR-PA) in the clinic.