This study shows that a cationic esterase mimic, which is responsible for the catalytic hydrolysis of a negatively charged fluorogenic ester, displays different inhibition behaviors in the presence of similar anionic metabolites in water and at physiological pH. On the contrary of the other negatively charged inhibitors tested, ADP strongly slowed down the formation of the fluorescent product by virtue of cooperative inhibition, hence allowing its spectroscopic or visual detection. Our sensor or its derivatives may find future use in biological applications, such as protein kinase activity detection.