In the present work, ZnO nanofibers were applied to electrode materials for the detection of cholesterol. ZnO nanofibers were synthesized using the electrospinning technique with zinc acetate as a precursor. Electrospinning-synthesized ZnO nanofibers were uniformly distributed by properly controlling the electrospinning parameters. After the calcination treatment, nanofibers of pure ZnO phase were synthesized. Then, these fibers were successfully placed on Au-coated glass substrates by dispersion of ZnO nanofibers in ethanol, dropping, and drying, in sequence. Cholesterol oxidase was then immobilized onto the surface of the ZnO nanofibers. To enhance the immobilization, Nafion was additionally applied. The sensing performances of the fabricated ZnO nanofibers-based sensors were analyzed by cyclic voltammetry in terms of cholesterol concentration ranging from 100 to 400 mg/dl. In the I-V curves, measured by cyclic voltammetry, the ZnO nanofiber-based sensor showed a proportional current behavior with cholesterol concentrations in phosphate buffered saline solution. The sensitivity was measured and found to be 30.7 nA/mM·cm2, which is comparable to the values reported in the literature. After not only optimizing the shape of the ZnO nanofibers but also improving the adhesion nature between the ZnO nanofibers and the Au conducting layer, these fibers can be a good candidate for electrode materials in devices used to detect low concentrations of cholesterol in blood.