Twisted bilayer graphene (tBLG) exhibits a host of innovative physical phenomena owing to the formation of moiré superlattice. Especially, the discovery of superconducting behavior has generated new interest in graphene. The growing studies of tBLG mainly focus on its physical properties, while the fabrication of high-quality tBLG is a prerequisite for achieving the desired properties due to the great dependence on the twist angle and the interfacial contact. Here, the cutting-edge preparation strategies and challenges of tBLG fabrication are reviewed. The advantages and disadvantages of chemical vapor deposition, epitaxial growth on silicon carbide, stacking monolayer graphene, and folding monolayer graphene methods for the fabrication of tBLG are analyzed in detail, providing a reference for further development of preparation methods. Moreover, the characterization methods of twist angle for the tBLG are presented. Then, the unique physicochemical properties and corresponding applications of tBLG, containing correlated insulating and superconducting states, ferromagnetic state, soliton, enhanced optical absorption, tunable bandgap, and lithium intercalation and diffusion, are described. Finally, the opportunities and challenges for fabricating high-quality and large-area tBLG are discussed, unique physical properties are displayed, and new applications inferred from its angle-dependent features are explored, thereby impelling the commercialization of tBLG from laboratory to market.