Over the last decades, cholinesterase (ChE) biosensors have emerged as a sensitive and rapid technique for toxicity analysis in environmental monitoring, food, and quality control. These systems have the potential to complement or replace the classical analytical methods by simplifying or eliminating sample preparation protocols and making field testing easier and faster with significant decrease in costs per analysis. In this study, a new bienzymatic biosensor based on butyrylcholinesterase (BuChE) and sulfhydryl oxidase (SOX) enzymes was developed. This system makes use of the biocatalyzed hydrolysis of butyrylthiocholine to butyric acid and thiocholine that acts as a SOX substrate. Measurements were performed by following of the consumed oxygen level related to butyrylthiocholine concentration in the enzymatic reactions. Bienzymatic system was characterized and applied for detection of donepezil which is a reversible inhibitor of cholinesterase and belongs to a new class of cholinesterase inhibitors having an N-benzylpiperidine and an indanone moiety, by following of the decrease in biosensor response as a result of competitive inhibition.