Butyrylcholinesterase (BChE) mainly contributing to plasma cholinesterase activity is an important indicator for routinely diagnosing liver function and organophosphorus poisoning in clinical diagnosis, but its current assays are scarce and frequently suffer from some significant interference and instability. Herein, we report a redox-controlled fluorescence nanoswtich based on reversible disulfide bonds, and further develop a fluorometric assay of BChE via thiol-triggered disaggregation-induced emission. Thiol-functionalized carbon quantum dots (thiol-CQDs) with intense fluorescence is found to be responsive to hydrogen peroxide, and their redox reaction transforms thiol-CQDs to nonfluorescent thiol-CQD assembly. The thiols inverse this process by a thiol-exchange reaction to turn on the fluorescence. The fluorescence can be reversibly switched by the formation and breaking of disulfide bonds caused by external redox stimuli. The specific thiol-triggered disaggregation-induced emission enables us to assay BChE activity in a fluorescence turn-on and real-time way using butyrylthiocholine iodide as the substrate. As-established BChE assay achieves sufficient sensitivity for practical determination in human serum, and is capable of avoiding the interference from micromolar glutathione and discriminatively quantifying BChE from its sister enzyme acetylcholinesterase. The first design of reversible redox-controlled nanosiwtch based on disulfide expands the application of disulfide chemistry in sensing and clinical diagnostics, and this novel BChE assay enriches the detection methods for cholinesterase activity.