In the current study, whey protein-based nanofibers were fabricated to encapsulate Lactobacillus rhamnosus. Purposely, different ratios of PVA (polyvinyl alcohol) and WPI (whey protein isolate) were blended to fabricate nanofibers. Nanofiber mats were characterized in terms of particle size, diameter, tensile strength, elongation at break, and loading efficiency. Morphological and molecular characterizations were carried out using scanning electron microscopy (SEM) and Fourier transform infrared (FTIR). Moreover, in vitro viability under simulated gastrointestinal (GI) conditions and thermal stability were also assessed. The results reveal that by increasing the PVA concentration, the conductivity increased while the viscosity decreased. SEM micrographs showed that probiotics were successfully loaded within the nanofiber. The FTIR spectra show strong bonding between the encapsulating materials with the addition of probiotics. In vitro and thermal analyses revealed that the survival of encapsulated probiotics significantly (p < 0.05) improved. In a nutshell, PVA–WPI composite nanofibers have promising potential when used to enhance the viability and stability of probiotics under adverse conditions.