Biofuel cells are energy conversion devices that use biocatalysts to convert chemical energy to electrical energy. Over the last decade, research in this area has intensified, especially in the area of direct electron transfer between enzymes and electrodes. This review details the fundamentals of enzymatic biofuel cells and reviews characterization techniques that can be used to evaluate and optimize biofuel cells. The review details the advantages and disadvantages of mediated and direct bioelectrocatalysis at electrodes for biofuel cells. It also compares and contrasts different enzyme immobilization techniques and different electrode structures.