The present chapter describes the use of biosensors based on enzyme inhibition as analytical tools. The parameters that affect biosensor sensitivity, such as the amount of immobilized enzyme, incubation time, and immobilization type, were critically evaluated, highlighting how the knowledge of enzymatic kinetics can help researchers optimize the biosensor in an easy and fast manner. The applications of these biosensors demonstrating their wide application have been reported. The objective of this survey is to give a critical description of biosensors based on enzyme inhibition, of their assembly, and their application in the environmental, food, and pharmaceutical fields.