The invention belongs to the fields of tissue engineering and a medical material, relates to an airway epithelium-porous silk fibroin protein complex, and a preparation method and application thereof, and particularly relates to a method for carrying out airway epithelium cultivation on porous silk fibroin protein, and application of the airway epithelium-porous silk fibroin protein complex in trachea defect reconstruction. The porous silk fibroin is adopted as a bracket material; a primary human airway epithelium cell is taken as a seed cell; the airway epithelium cell is subjected to composite cultivation on the porous silk fibroin protein, so as to form the airway epithelium-porous silk fibroin protein complex. The complex is covered on the inner wall of an artificial trachea, so that the inner wall of the artificial trachea is covered by respiratory mucosa, so as to be closer to a normal trachea structure; the artificial trachea can be further applied to preparation of a tissue engineering trachea, repair of clinical tracheal mucosa and clinical tracheal defect reconstruction, and reduction of postoperative complications of tracheal reconstruction as the medical material.