Biofuel cells provide a versatile means to generate electrical power from environmentally friendly biomass or biofuels. Immobilization technology has played an important role in the design of biofuel cells. This review addresses recent advances in immobilization technology applied to assembling biofuel cells. After identifying the advantages and problems of biofuel cells, the immobilization technology, which offers a sustainable and effective solution for biofuel cell design, is thoroughly presented: (i) A brief introduction to immobilization methods, including adsorption, covalent binding and entrapment, is first presented. (ii) The immobilization structure and nanostructures are emphasized, which strongly influence mass and electron transfer, including zero, one, two, and three dimensional nanostructures are then discussed. (iii) The immobilization materials, which are considered as a critical factor in immobilization technology, including polymer, carbon, oxide and metallic nanomaterials, sol–gel based materials and particularly composite materials, are reviewed to conclude. The interesting issues related to the future of biofuel cell design are also highlighted, for example, the 3D electrode assembly using low dimensional structures as a future challenge, biofuel cells within logic systems as a new aspect, crystalline mesoporous carbon with high enzyme loading as a future desired material, and composite materials with multiple functions and structures as a hot area of work.