The invention particularly relates to a double-layer artificial periosteum, a preparation method and application thereof, and belongs to the technical field of biomedical materials. The double-layer artificial periosteum is characterized in that a compact layer is prepared firstly, and then a loose layer is prepared on the compact layer. According to the invention, an artificial periosteum with a double-layer structure is constructed, wherein a compact layer and a loose layer are synergistically combined, the surface of the compact layer is smooth, the compact layer faces soft tissue during use, the physical barrier function of the periosteum can be well utilized, connective tissue is effectively prevented from growing into a bone defect area, and the regeneration function of specific tissue is played to the maximum extent; the coarse loose layer is consistent with the autogenous bone in component and structure, faces bone tissue during use, can be well attached to a bone defect surface, induces generation of new bones, effectively inhibits osteosarcoma and treats bone cancer; and the obtained double-layer artificial periosteum has good mechanical properties and degradation properties, and also has good capabilities of promoting bone healing, inhibiting osteosarcoma and treating bone cancer.