The review summarizes the fabrication, microstructures, and sensor applications of highly ordered electrospun nanofibers. In the traditional electrospinning process, electrospun nanofibers usually have disordered or random microstructures due to the chaotic oscillation of the electrospinning jet. Different electrospinning methods can be formed by introducing external forces, such as magnetic, electric, or mechanical forces, and ordered nanofibers can be collected. The microstructures of highly ordered nanofibers can be divided into three categories: uniaxially ordered nanofibers, biaxially ordered nanofibers and ordered scaffolds. The three microstructures are each characterized by being ordered in different dimensions. The regulation and control of the ordered microstructures can promote electrospun nanofibers’ mechanical and dielectric strength, surface area and chemical properties. Highly ordered electrospun nanofibers have more comprehensive applications than disordered nanofibers do in effect transistors, gas sensors, reinforced composite materials and tissue engineering. This review also intensively summarizes the applications of highly ordered nanofibers in the sensor field, such as pressure sensors, humidity sensors, strain sensors, gas sensors, and biosensors.