Nanofibers fabricated via electrospinning have drawn much attention nowadays due to its large surface area, and easy production of nanoscaled structure for interaction with biomolecules. This chapter presents an overview for the recent applications of nanofibers as scaffolds for enzyme immobilization. This chapter contains a) brief introduction to conventional nanofiber production via electrospinning, b) recent application of nanofibers as scaffolds for enzyme immobilization and c) its application to biocatalytic system. Nanometer-sized materials are of considerable interest in analytical chemistry and biotechnology due to their potential applications in bioassay, medical diagnostics, and catalysis. The recent advances in the creation of new nanomaterials and nanoscaled devices trigger off the new direction of biological research ( / ). The nanofibers for the application in many biotechnology areas have drawn much attention nowadays. Depending on polymer materials used, various surface functionalized nanofibers could be produced. In this chapter, an overview on the recent application of nanofibers for biological applications, especially as scaffolds for enzyme immobilization, is presented.