Nanofibers consisting of enzyme-polymer composites have been prepared by directly electrospinning a solution of surfactant-stabilized enzyme and polymer in toluene. Additional treatment with glutaraldehyde could greatly stabilize the enzyme activity of the fibers, which could be maintained in a buffer under shaking conditions for more than two weeks. The nanofibers also showed great improvement in the enzyme activity over bulk films as a result of increased mass-transfer for substrate molecules to and from the enzyme reactive sites. These stable and catalytically active nanofiber-based mats were highly durable and could be easily recovered from solution, making them ideal candidates for large scale applications.