Biosensors are available and applicable for detection and characterization of specific inhibitors of many enzymes. In this review, biosensors based on fixed acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) are presented. Inhibition of selected enzymes by various compounds, such as organophosphorus and carbamate pesticides, nerve agents (e.g. sarin or VX), and other natural toxins (e.g. aflatoxins), was employed to develop specific assays using biosensors only. Biosensor technology brings potential miniaturization and portability, when it is compared to standard methods. Construction of biosensors based on cholinesterases became a more important issue within the last decades. Novel approach with recombinant proteins, microelectrodes and immobilization protocol related to nanotechnologies opened new insight to the cholinesterase based biosensor construction and its perspective via routine assays. This review is focused on novel trends within such biosensors as a result of the known platform.