: Since the first glucose biosensor was developed by Clark and Lyons, there have been great efforts to develop effective enzyme biosensors for wide applications. Those efforts are closely related to the enhancement of biosensor performance, including sensitivity improvement, elevation of selectivity, and extension of the range of analytes that may be determined. Introduction of a cascadic multienzyme reaction to the electrochemical biosensor is one of those efforts. By employing more than two enzymes to the biosensor, its sensitivity and accuracy can be enhanced. Also, the narrow application range that is a typical limitation of single enzyme-based biosensor can be overcome. This chapter will discuss the fundamental principles for the development of cascadic multienzyme reaction-based electrochemical biosensors and their applications in clinical and environmental fields.