The authors describe a method for the preparation of orange-red emissive carbon dots (CDs) with excitation/emission peaks at 520/582 nm. The CDs were hydrothermally prepared by a one-pot strategy from trimesic acid and 4-aminoacetanilide. The fluorescence of the CDs is strongly quenched by hydrogen peroxide. The oxidation of glucose by glucose oxidase (GOx) produces H2O2 that quenches the fluorescence via static quenching. Based on this phenomenon, a fluorometric method was established for the determination of glucose. Under the optimum conditions, response is linear in the 0.5 to 100 μM glucose concentration range, with a 0.33 μM limit of detection. The method is selective for glucose over its analogues and was successfully applied to the determination of glucose in diluted human serum and in urine from diabetics and healthy individuals. Recoveries from spiked samples range from 98.7 to 102.5%.