Analytical systems integrating in vivo microdialysis sampling with enzyme-based electrochemical biosensor detection have been increasingly accepted to be a new technique for continuous and on-line monitoring of biologically important species. Extensive interests in such integrated on-line analytical systems have suggested that these systems are very useful for physiological and pathological investigations. This review mainly focuses on the principle, development and striking applications of the enzyme-based amperometric biosensors integrated with in vivo microdialysis for continuous and on-line monitoring of cerebral extracellular fluid in recent years.