An enzyme biosensor is an investigative device that joins an enzyme with a transducer to create a signal corresponding to target analyte fixation. An ideal enzyme activity is a basis for the maintenance of physiological homeostasis of biosensor. Both non-hereditary and hereditary disturbances can too much initiate or quiet characteristic enzyme activities. Due to its virtuous sensitivity, easy to operate, high precision and low instrumentation cost it can be used for recognition of various analytes in different fields than the traditional analytical methods. The enzyme based biosensor is most commonly used dominant tool for the determination of various biological importance such as antigens, antibodies, therapeutic drugs and metabolites. In regular enzymebased biosensors, signal amplification is not satisfactory for the ultrasensitive identification of biomolecules and it was enhanced by consolidating enzymatic responses with redox cycling of multi-enzyme tags for every discovery test. This chapter explains the working principles, features, types, sensing methods and applications of enzyme based biosensor in various fields.