In the current study, the probiotic (Lactobacillus acidophilus) was encapsulated using Gum Arabic and polyvinyl alcohol blended nanofibers by electrospinning. Obtained nanofibers were characterized in terms of particle size, diameter, mechanical strength, and encapsulation efficiency. The molecular and internal structure characterization was carried out using Fourier transform infrared spectroscopy and X-ray diffraction respectively. Thermo Gravimetric (TGA) analysis was conducted to determine the thermal features of PVA/GA/probiotics nanofibers. Free and encapsulated probiotics were also subjected to in vitro assay under different detrimental conditions. Images obtained using SEM indicated that probiotics were successfully encapsulated in blends by a nano-spider. FTIR and XRD spectra showed bonding interactions between the wall and core materials. In-vitro assay indicated that probiotics with encapsulated showed significantly (P