The invention discloses chitosan nanofiber aligned by an electric field and a preparation method and application thereof. The preparation method comprises the following steps: (1) chitosan is dissolved with acid to obtain a dilute chitosan solution; (2) the dilute chitosan solution is placed into an electric field; low-temperature phase separation is carried out under the circumstance of staying energized; and then freeze-drying is conducted to obtain the chitosan nanofiber aligned by the electric field. The preparation method has the following advantages: the process is simple and feasible, can be combined with a three-dimensional scaffold material with a macroporous structure, and has high flexibility and adaptability. The prepared chitosan nanofiber has a regular alignment structure, achieves bionic construction of a natural extracellular matrix in structural performance, and has a wide application prospect in the fields of tissue engineering materials and biomimetic materials.