A parallel G-quadruplex-selective iridium(III) complex has been synthesized and employed as a lumines-cent probe in a label-free G-quadruplex-based detection assay for Ca2+ions in aqueous solution. In thisassay, a guanine-rich oligonucleotide (G4, 50-G4T4G4-30) initially exists in an antiparallel G-quadruplexconformation, resulting in a low luminescence signal. Upon incubation with Ca2+ions, the antiparallelG-quadruplex is induced into a parallel G-quadruplex conformation, which greatly enhances the lumi-nescence emission of the iridium(III) probe. This method was highly sensitive for Ca2+ions with a limitof detection in the nanomolar range, and was selective for Ca2+over other metal ions.