A label-free oligonucleotide-based luminescent switch-on assay has been developed for the selective detection of sub-nanomolar Pb²⁺ ions in aqueous solution and real water samples. An iridium(III) complex was employed as a G-quadruplex specific luminescent probe and a guanine rich DNA (PS2.M, 5′-GTG₃TAG₃CG₃T₂G₂-3′) was employed as recognition unit for Pb²⁺ ions. The PS2.M exists in a single-stranded conformation in the absence of Pb²⁺ ions, and the weak binding of the iridium(III) probe to ssDNA results in a weak luminescence signal. Upon binding to Pb²⁺ ions, the single-stranded DNA sequence (PS2.M) is induced into a G-quadruplex conformation, which greatly enhances the luminescence emission of the iridium(III) probe. The assay can detect Pb²⁺ ions in aqueous media with a limit of detection of 600 pM. It also exhibits good selectivity for Pb²⁺ ions over other heavy metal ions. Furthermore, the application of the assay for the detection of Pb²⁺ ions in spiked river water samples has been demonstrated.