Analytical technology based on sensors is an extremely broad field which impacts on many major industrial sectors such as the pharmaceutical, healthcare, food, and agriculture industries as well as environmental monitoring. This review will highlight the research carried out during the last 5 years on biosensors that are based on enzyme inhibition for determination of pollutants and toxic compounds in a wide range of samples. Here the different enzymes implicated in the inhibition, different transducers forming the sensing devices, and the different contaminants analyzed are considered. The general application of the various biosensors developed, with emphasis on food and environmental applications, is reviewed as well as the general approaches that have been used for enzyme immobilization, the enzyme catalysis, and the inhibition mechanism.