As an advanced biosensing strategy, the electrochemical biosensor is made up of the active sensing biomaterial and the electrochemical signal transducer, and is being widely applied in the fields of clinical medicine, drug and food analysis as well as the environmental monitoring. The electrochemical biosensor has the advantages of excellent specificity, high sensitivity and simplicity. This review focused on the fundamental principle of electrochemical biosensor, its classification and the biomedical applications. The prospect of the electrochemical biosensors was outlined as well.