Uracil-deoxyribonucleic acid glycosylase (UDG) is known to function as an important base-excision repair enzyme and eliminate uracil from DNA molecules to maintain genomic integrity. A new small organic molecule (DID-VP) with interesting structural properties was synthesized as a G-quadruplex selective ligand and was demonstrated to be a sensitive luminescent switch-on probe in a convenient luminescent assay specifically for UDG detection in fetal bovine serum samples under rapid and simple conditions. This newly developed analytical method is based on the UDG enzymatic activity to unwind a duplex DNA substrate, and comprises a G-quadruplex-forming sequence (ON1) and uracil-containing DNA strand (ON2) to generate a remarkable fluorescence signal through the specific interaction of DID-VP with ON1. This luminescent switch-on assay is able to achieve high sensitivity and specificity for UDG over other enzymes. The application range of the present analytical system is found to be 0.05 to 1.00 U mL⁻¹ UDG with a very low detection limit of 0.005 U mL⁻¹. The recovery study of UDG in real samples gave a very good performance with 75.05%–102.7% recovery. In addition, an extended application of the assay in screening of UDG inhibitors is demonstrated. A good dose-dependence of the luminescence response with respect to the concentration of UDG inhibitors in samples was observed. Copyright © 2015 The Royal Society of Chemistry.