A series of luminescent Ir(iii) complexes were synthesised and evaluated for their ability to act as G-quadruplex-selective probes. A novel Ir(iii) complex was found to be highly selective for G-quadruplex DNA, and was employed in a label-free G-quadruplex-based detection assay for nicking endonuclease activity in aqueous solution. A proof-of-concept of this probe has been demonstrated by using Nb·BbvCI as a model enzyme. In this assay, a DNA substrate comprised of oligonucleotides ON1 (5′-GTG3TAG3CG3T2G2CTGAG2TGA-3′) and ON2 (5′-TCAC2TCAGC2A2C2-3′) initially exists in a duplex conformation, resulting in a low luminescence signal due to the weak interaction between the Ir(iii) complex and duplex DNA. Upon cleavage by Nb·BbvCI, the guanine-rich sequence is released and folds into a G-quadruplex, which greatly enhances the luminescence of the Ir(iii) probe. This method was highly sensitive for Nb·BbvCI over other DNA-modifying enzymes. This journal is © the Partner Organisations 2014.