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For improving probiotics’ survivability under harsh conditions, this study used <i>Lactiplantibacillus plantarum</i> GIM1.648 as a model microorganism to investigate its ability to produce biofilms on electrospun ethyl cellulose nanofiber mats. SEM observations confirmed that biofilm was successfully formed on the nanofibers, with the latter being an excellent scaffold material. The optimal cultivation conditions for biofilm formation were MRS medium without Tween 80, a culture time of 36 h, a temperature of 30 °C, a pH of 6.5, and an inoculum concentration of 1% (<i>v/v</i>). The sessile cells in the biofilm exhibited improved gastrointestinal and thermal tolerance compared to the planktonic cells. Additionally, the RT-qPCR assay indicated that the <i>luxS</i> gene played a crucial role in biofilm formation, with its relative expression level being 8.7-fold higher compared to the planktonic cells. In conclusion, biofilm formation on electrospun nanofiber mat has great potential for improving the viability of probiotic cells under harsh conditions.