Various methods of immobilizing enzymes on electrodes have been developed for the research of enzymatic biofuel cells (EBFCs). Among these methods, the layer-by-layer (LbL) assembly is a delicate technology that can form controllable enzyme layers and load a high density of enzyme molecules by electrostatic deposition, hydrogen bonding, or covalent bonding. In this paper, recent advancements of the LbL assembly for immobilizing enzymes in EBFCs are presented in detail. First, the LbL formation mechanism of a multilayer sensitive film on electrodes is analyzed, and the potential of the multilayer structure is also discussed. Then, the applications of enzyme immobilization by the LbL assembly in combination with various materials in EBFCs are investigated. The power density of LbL assembly-based EBFCs increases by several orders of magnitude with the widespread use of nanomaterials. As a convenient and efficient operation, the LbL assembly technology is expected to become a popular method for immobilizing enzymes in EBFCs.