Glibenclamide (GB), as a sulfonylurea-based medication is commonly prescribed for the treatment of type 2 diabetes. Due to its increasing consumption, there is a need to develop a simple, fast, and reliable detection method to follow its concentration in pharmaceutical and biological samples. Herein, a novel fluorometric method is developed for the sensitive measurement of GB. The method is based on the enhancing effect of GB on the fluorescence emission of mercaptopropionic acid (MPA) capped cadmium telluride quantum dots (CdTe QDs). QDs were synthesized in aqueous solution and were characterized by fluorescence spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR). Fluorescence intensity of QDs was enhanced by adding GB in a very low concentration. The effect of operative factors such as pH, buffer, contact time and concentration of CdTe QDs were investigated and in the optimized condition, a linear increase was achieved for the emission intensity of QDs by increasing GB concentration in the range 49-345 ng mL −1 , with a detection limit of 17.84 ng mL −1 . The offered method has an acceptable precision (relative standard deviations were < 2.8%) and was satisfactorily applied for the determination of GB in pharmaceutical products and human urine samples.