To explore the resistance mechanism of high biofilm production in Lactobacillus fermentum 733， its tolerance to simulated artificial gastrointestinal fluids， acid， bile salts， temperature， and salt stress， as well as the secretion of harmful metabolites， were determined. Additionally， a whole-genome sequencing analysis was conducted. The results showed that the survival rates of high biofilm production L. fermentum 733 in simulated artificial gastrointestinal fluids were (98.1±0.5)% and (98.1±1.2)%， respectively， indicating high survival rates， and the strain did not produce harmful metabolites. Growth of the strain was inhibited in different stress environments， while under specific conditions， it stimulated stress responses to ensure strain vitality. The L. fermentum 733 mainly affected the trxA， perR， ctsR and argR genes when experiencing different environmental stresses， however， how these genes regulate the stress resistance of strain remains to be further studied.