Abstract: In order to integrate the natural biofilm of probiotics with microencapsulation technology in a synergistic manner, so as to construct a novel and synergistic probiotic protection strategy. This study aimed to select the optimal combination of bacterial strains from various prebiotic culture media based on their growth rate and ability to form biofilms. The selected strains were then used to prepare single-layer, double-layer, biofilm-like single-layer, and biofilm-like double-layer microcapsules. The protective effects of the freeze-dried microcapsules containing the selected strains on the viability of the probiotics under simulated gastrointestinal conditions and conditions of low temperature storage were investigated. The results showed that the highest growth and biofilm formation was achieved when Lactobacillus fermentum F7 and Bifidobacterium longum B22 were co-cultured in a medium containing 1.5% IMO at a 1:1 inoculation ratio. The particle size was determined to be in the range 2.29~2.45 mm for all four microcapsule types, and viable counts were recorded between 9.58~10.03 lg CFU/g. Stress tests showed that the biofilm-like bilayer microcapsules provided the strongest protection against the compound bacteria, with a survival rate of more than 93% after freeze-drying. Even after storage at −18 ℃ and 4 ℃ for 7 weeks, the viable counts were maintained above 10⁸ CFU/g in the biofilm-like bilayer microcapsules. This study lays the foundation for the development of freeze-dried biofilm-like composite probiotic microcapsules and suggests an effective protective strategy for probiotics under stressful conditions.