We report herein the synthesis and application of a novel G-quadruplex-selective luminescent iridium(III) complex [Ir(ppy)2(bcp)](+) (where ppy = 2-phenylpyridine and bcp = 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline) for the sensitive detection of apurinic/apyrimidinic (AP) endonuclease activity. Using endonuclease IV (Endo IV) as a model enzyme, a duplex DNA substrate containing a G-quadruplex-forming sequence is cleaved by Endo IV at the abasic site. This releases the G-quadruplex sequence, which folds into a G-quadruplex and is recognised by the G-quadruplex-selective iridium(III) complex with an enhanced luminescence response. The assay achieved high sensitivity and selectivity for Endo IV over other tested enzymes.