Biofuel cells refer to a class of fuel cells that apply biocatalysts to power generation. Early observations of electricity generation from glucose and other organic compounds in the presence of microbes dates back to the early 1900s, while the growing interest in searching for bio-based alternative energy resources has spurred a new wave of biofuel cell development in recent years. Compared with metal catalyst-based traditional fuel cells, biofuel cells involve very different reaction routes with water as an essential reaction medium, therefore affording a mild operation condition, the use of biofuels and the application of newly emerged materials such as carbon nanotubes. Current research in this area has mostly focused on realizing the best potential of such advantages, and the overall performance of biofuel cells are steadily improving. This paper reviews the evolution and development of biofuels and biocatalysts for biofuel cells gleaned from recent publications, and presents an overview and discussion on factors limiting the power-generation efficiency of biofuel cells.