The creation of complex multilayer periosteal graft structures is challenging. This study introduced a novel bottom-up approach to assemble cell-laden nanofiber mats into a threedimensional (3D) multilayer periosteum mimic, successfully replicating the hierarchical complexity of the natural periosteum. These nanofiber mats, which were fabricated by electrospinning, surface modification, and stimulated body fluid (SBF) immersion, are composed of nanoscale polycaprolactone (PCL) fibers coated with a mineralized collagen layer along the fiber orientation. They closely resembled the natural periosteal matrix, thereby promoting osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. The biomimetic periosteum, constructed via layer-by-layer assembly, offered advantages such as a multilayer nanofibrous structure, controlled cell distribution, a reservoir for osteoprogenitors, and enhanced pro-osteogenic potential. The rat calvarial bone defect model confirmed its potent bone repair capacity. This study presents an efficient approach to construct tissueengineered periosteum mimics, holding promise for serving as periosteal grafts in orthopedic applications.