The development of glucose biosensors has been attracting much attention because of their importance in monitoring glucose in the human body; such sensors are used to diagnose diabetes and related human diseases. Thanks to the high selectivity, sensitivity to glucose detection, and relatively low-cost fabrication of enzyme-immobilized electrochemical glucose sensors, these devices are recognized as one of the most intensively investigated glucose sensor types. In this work, ZnO nanofibers were synthesized using an electrospinning method with polyvinyl alcohol zinc acetate as precursor material. Using the synthesized ZnO nanofibers, we fabricated glucose biosensors in which glucose oxidase was immobilized on the ZnO nanofibers. The sensors were used to detect a wide range of glucose from 10 to 700 M with a sensitivity of 10.01 nA/cm2-μM, indicating that the ZnO nanofiber-based glucose sensor can be used for the detection of glucose in the human body. The control of nanograins in terms of the size and crystalline quality of the individual nanofibers is required for improving the glucose-sensing abilities of the nanofibers.