Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are enzymes expressed in the human body under physiological conditions. AChE is an important part of the cholinergic nerves where it hydrolyses neurotransmitter acetylcholine. Both cholinesterases are sensitive to inhibitors acting as neurotoxic compounds. In analytical applications, the enzymes can serve as a biorecognition element in biosensors as well as simple disposable sensors (dipsticks) and be used for assaying the neurotoxic compounds. In the present review, the mechanism of AChE and BChE inhibition by disparate compounds is explained and methods for assaying the enzymes activity are shown. Optical, electrochemical, and piezoelectric biosensors are described. Attention is also given to the application of sol-gel techniques and quantum dots in the biosensors’ construction. Examples of the biosensors are provided and the pros and cons are discussed.