All the disciplines of science, especially biotechnology, have given continuous attention to the area of enzyme immobilization. However, the structural support made by material science intervention determines the performance of immobilized enzymes. Studies have proven that nanostructured supports can maintain better catalytic performance and improve immobilization efficiency. The recent trends in the application of nanofibers using natural polymers for enzyme immobilization have been addressed in this review article. A comprehensive survey about the immobilization strategies and their characteristics are highlighted. The natural polymers, e.g., chitin, chitosan, silk fibroin, gelatin, cellulose, and their blends with other synthetic polymers capable of immobilizing enzymes in their 1D nanofibrous form, are discussed. The multiple applications of enzymes immobilized on nanofibers in biocatalysis, biosensors, biofuels, antifouling, regenerative medicine, biomolecule degradation, etc.; some of these are discussed in this review article. Strategies of immobilization of enzymes using natural polymeric nanofibers have been described in this review. Khan and coworkers have presented an overview of the immobilization of enzymes on nanofibers fabricated via electrospinning which provides multiple ways of stabilization of enzymes on a substrate for different biological applications. The authors have discussed the properties of natural polymers used as supports for enzyme immobilization and have presented a comprehensive discussion of the work done in this area for biocatalysis, biosensors, and biofuels.