Nanofibers have many promising applications as a main component of one-dimensional nanomaterials. Electrospinning technology is a facile and effective way to prepare nanofibers. However, the traditional preparation process only obtains non-aligned nanofibers, which significantly hinders their application. In recent few decades, many efforts have been focused on optimization of the preparation method, and well aligned nanofibers (ANFs) have been successfully obtained through improving spinning set-ups, collectors, and optimizing the separation section of nanofibers. Due to the absence of a systematic appraisal of ANFs, here a review of the preparation methods of ANFs based on electrospinning and their applications in tissue engineering regeneration, sensors, reinforced materials, sensors and energy devices, is present. Owing to the wide application of ANFs in tissue engineering regeneration, a detailed discussion is provided in that regard. In the field of energy, the application of ANFs in PEM fuel cells is focused. Finally, the challenges and outlooks for the development of ANFs are summarized.