Abstract: The objective of this study was to reveal the difference of biofilm formation of Bifidobacterium on insoluble dietary fibers under dynamic cultivation,and characterize the resistance of biofilms. Based on the viable cell count and structural characterization of biofilms,it was found that the grape seed fiber was the most suitable carrier for biofilm formation among the six different types of insoluble dietary fibers,followed by wheat fiber. The optimal biofilm-forming time of Bifidobacterium was investigated. The optimal biofilm formation time based on the grape seed fiber was 36 hours,while the optimal biofilm formation time based on wheat fiber was 24 hours. The experiment further compared the differences between the biofilm-forming cells and the planktonic cells on the survival rate of freeze-drying and simulated gastrointestinal fluid tolerance. The biofilm cells exhibited higher freeze-drying resistance(4~6.5×). The survival rate of simulated gastric juice was increased by 5.1-5.9 times(P<0.01),and the highest survival rate reached to 59.82%±2.32%. The survival rate of simulated intestinal fluid was increased by 10-12.6 times(P<0.01),and the highest survival rate reached to 13.53%±1.5%. These results indicated that although Bifidobacterium had different biofilm-forming capabilities on different insoluble dietary fibers,while the stress resistance could be significantly improved after biofilm formation. The research results provide a theoretical basis for the application of the food industry in the development of stress-resistant probiotics.