In this study, an unreported Ir(III) complex 1 was identified by screening as a versatile G-quadruplex probe. It exhibited highly selective response for different G-quadruplex DNA over double strand, single strand and triplex DNA. Compared with the organic G-quadruplex probe thioflavin T, complex 1 displays a longer lifetime, a larger Stokes shift, comparable G-quadruplex/ssDNA enhancement ratios, and higher G-quadruplex/triplex DNA enhancement ratios. In consideration of the encouraging G-quadruplex probe performance of complex 1, we employed 1 to develop a G-quadruplex-based detection system for the detection of insulin as a "proof-of-principle" concept. We also demonstrate an optimization process that enhanced the sensitivity of this sensing assay. Compared to previously reported methods, our "mix-and-detect" detection methodology is easy operated, quick, and cost-effective. A detection limit as low as 80 pM for insulin can be achieved by this sensing approach, with a linear relationship between luminescence intensity and insulin concentration established from 80 pM to 20 nM. Moreover, this assay could work effectively in diluted human serum.