Polyacrylonitrile/polyurethane/poly(m-anthranilic acid) nanofibers were fabricated by electrospinning. Tyrosinase immobilization was performed by EDC/N-hydroxyl succinimide activation. Covalent binding of tyrosinase onto nanofibers was confirmed by Fourier transform infrared-attenuated total reflectance, and bicinchoninic acid assay revealed the amount of enzyme. Nanofiber morphology and composition were characterized by scanning electron microscopy/energy-dispersive X-ray spectroscopy (EDX). Nanofibers became smoother and thicker after tyrosinase immobilization. Effects of enzyme on nanofibers were investigated by electrochemical impedance spectroscopy and the data were fitted to equivalent electrical circuit model. EDX-mapping showed uniform distribution of enzyme. The solution resistance and charge transfer resistance of nanofibers decreased after enzyme immobilization.