Diabetes is a chronic, systemic metabolic disease that leads to multiple complications, even death. Meanwhile, the number of people with diabetes worldwide is increasing year by year. Sensors play an important role in the development of biomedical devices. The development of efficient, stable, and inexpensive glucose sensors for the continuous monitoring of blood glucose levels has received widespread attention because they can provide reliable data for diabetes prevention and diagnosis. Electrospun nanofibers are new kinds of functional nanocomposites that show incredible capabilities for high-level biosensing. This article reviews glucose sensors based on electrospun nanofibers. The principles of the glucose sensor, the types of glucose measurement, and the glucose detection methods are briefly discussed. The principle of electrospinning and its applications and advantages in glucose sensors are then introduced. This article provides a comprehensive summary of the applications and advantages of polymers and nanomaterials in electrospun nanofiber-based glucose sensors. The relevant applications and comparisons of enzymatic and non-enzymatic nanofiber-based glucose sensors are discussed in detail. The main advantages and disadvantages of glucose sensors based on electrospun nanofibers are evaluated, and some solutions are proposed. Finally, potential commercial development and improved methods for glucose sensors based on electrospinning nanofibers are discussed.