Recent progress in reagentless electrochemical biosensors is reviewed. Electrochemical biosensors are classified into two categories, mediated and mediatorless, and will be accordingly discussed in this review. Special attention is paid to the way to direct electron transfer of enzymes, which forms the basis of third generation electrochemical biosensors. Different immobilizing approaches of enzymes at solid electrode surfaces are described. Their applications in biochemical research, clinical detection and environmental protection are addressed.