The nervous system regulates the visual system through neurotransmitters that play an important role in visual and ocular functions. One of those neurotransmitters is acetylcholine, a key molecule that plays a variety of biological functions. Moreover, acetylcholinesterase, the enzyme responsible for the hydrolysis of acetylcholine, is implicated in cholinergic function. However, several studies have demonstrated that in addition to their enzymatic functions, acetylcholinesterase exerts non-catalytic functions. In recent years, the importance of evaluating all possible functions of acetylcholine-acetylcholinesterase has been shown. Nevertheless, there is evidence suggesting that cholinesterase activity in the eye can regulate some biological events both in structures of the anterior and posterior segment of the eye and, therefore, in the visual information that is processed in the visual cortex. Hence, the evaluation of cholinesterase activity could be a possible marker of alterations in cholinergic activity in both ocular and systemic diseases.