Oral fast dissolving films represent a novel dosage form for probiotics. To reduce the dependence of film preparation on synthetic materials, a polysaccharide-based oral fast dissolving nanofilm for probiotics was fabricated through pullulan (PUL) electrospinning. An electrospinnability map of PUL with varying physical properties was developed, identifying a molecular weight of 200 kDa and a concentration of 20 % as suitable conditions for achieving favorable fiber morphology. Scanning electron microscopy, Fourier-transform infrared spectroscopy, and fluorescence assays confirmed that probiotics could be effectively encapsulated in the nanofilm, with 92.6 % of viable cells retained after electrospinning. Results of thermogravimetric analysis and thermal test indicated that the heat resistance of the encapsulated bacteria was significantly improved (P <
0.05). After 28 days of storage, the loss of viable bacteria was higher at 25 °C (2.9 log) than at 4 °C (0.5 log). This observation is consistent with the results of accelerated storage test, which showed that probiotic nanofilms stored at 4 °C had a longer shelf life with an inactivation rate constant of 1.74 × 10