Sialic acid (Sia) binding immunoglobulin (Ig)-like lectin-5 (Siglec-5) is a type-I transmembrane protein, and it has been demonstrated as a biomarker of granulocytic maturation and acute myeloid leukemia phenotype. Herein we aimed to construct a method that could sensitively detect Siglec-5 by taking advantage of the high affinity and selectivity of the K19 aptamer for its cognate target, and the selective interaction of luminescent iridium(III) transition metal complexes with G-quadruplex DNA. The iridium(III) complex 1 [Ir(tpyd)(2,9-dmphen)]PF (where tpyd =2-(m-tolyl)pyridine; 2,9-dmphen =2,9-dimethyl-1,10-phenanthroline) was synthesized, and it displayed high luminescence for G-quadruplex DNA compared to dsDNA and ssDNA. Additionally, complex 1 exhibited a blue shift luminescence response to c-kit2 G-quadruplex, and the interaction between 1 and G-quadruplexes was discussed based on the results of G-tetrad assay, loop effect assay, and other assays. Then complex 1 was utilized to develop a G-quadruplex-based sensing platform for Siglec-5 in aqueous solution. Upon the addition of Siglec-5, the specific binding of the K19 aptamer sequence results in a conformational change that generates a split G-quadruplex structure, which is then recognized by the G-quadruplex-specific iridium(III) complex with an enhanced luminescent response. Futhermore, the use of the assay for detecting Siglec-5 in cellular debris was demonstrated.