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Biofilm bacteria have stronger resistance to the adverse external environment compared to planktonic bacteria, and biofilms of non-pathogenic bacteria have strong potential for applications in food. In this experiment, Halomonas sp. H09 and Lactobacillus rhamnosus GG, which have film-forming ability in monoculture and better film-forming ability in mixed culture than the two strains alone, were selected as the target strains for mixed culture. According to SEM observation and bacterial dry weight measurement, the target strain formed a dense biofilm on a 0.1 g/L chitosan-modified cellulose III carrier. Furthermore, the presence of extracellular polymeric substances in biofilms was verified by EDS and FTIR. The results showed that 0.1 g/L chitosan-modified cellulose III was an ideal carrier material for immobilization of Halomonas sp. H09 with Lactobacillus rhamnosus GG biofilm. This research provided a basis for the selection of non-pathogenic mixed-bacteria biofilm carriers., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)