A panel of six luminescent iridium(III) complexes were synthesized and evaluated for their ability to act as G-quadruplex-selective probes. The novel iridium(III) complex 1 was found to be highly selective for G-quadruplex DNA, and was employed for the construction of a label-free G-quadruplex-based adenosine detection assay in aqueous solution. Two different detection strategies were investigated for adenosine detection, and the results showed that initial addition of adenosine to the adenosine aptamer gave superior results. The assay exhibited a linear response for adenosine in the concentration range of 5 to 120 μM (R 2 = 0.992), and the limit of detection for adenosine was 5 μM. Moreover, this assay was highly selective for adenosine over other nucleosides, and exhibited potential use for biological sample analysis.