Thymine DNA glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation, which also plays an essential role in DNA demethylation. In this work, the novel iridium(III) complex 1 with an anchor tail was synthesized and employed to construct a G-quadruplex-based assay for detecting TDG activity in aqueous solution by using the mismatched base excising property of TDG with T4 DNA ligase and phi29 DNA polymerase, in concert with the rolling circle amplification (RCA) strategy. The assay achieved a detection limit of 0.048 U mL (0.012 ng mL), and showed high selectivity towards TDG even in the presence of other proteins and enzymes. Additionally, the assay could function in diluted cellular debris.