Biotechnological use of probiotic microorganisms involves providing them with appropriate conditions for growth, but also protection against environmental changes caused by an exchange of the medium, isolation of metabolites, etc. Therefore, the research on effective immobilization of probiotic microorganisms should be focused in this direction. The present study aimed to evaluate the effectiveness of an innovative hybrid immobilization system based on electrospun nanofibers and alginate hydrogel. The analyses carried out included the study of properties of the initial components, the evaluation of the degree and durability of cell immobilization in the final material, and their survival under stress conditions. Effective binding of microorganisms to the hydrogel and nanofibers was confirmed, and the collected results proved that the proposed biocomposite is an efficient method of cell protection. In addition, it was shown that immobilization on electrospun nanofibers leads to the preservation of the highest cell activity and the least cell growth restriction as compared to free or lyophilized cells only. The completed research opens new perspectives for the effective immobilization of microorganisms of significant economic importance.