ABSTRACT This work reported a label-free single hairpin DNA-based self-propelled dual-cycle targets recycling strategy for ultra sensitive detection of Hg2+ based on T-Hg2+-T structure, exonuclease Ш (Exo Ш)-assisted self-propelled dual-cycle targets recycling and haemin/G-quadruplex (DNAzyme) signal amplification. The hairpin DNA probe with G-quadruplex fragment and continuously repeated sequences was specially designed for this strategy. Hg2+ could assist the combination between Hg2+ binding probe and free 3ʹ-terminus of hairpin DNA probe by T-Hg2+-T coordination to form the blunt 3ʹterminus that triggered Exo III-assisted digestion, and set free Hg2+, Hg2+ binding probe, and free G-quadruplex fragment. The released Hg2+ and Hg2+ binding probe is recycled through cycle 1. The released free G-quadruplex fragments are recycled through cycle 2. Single hairpin DNA-propelled dual-cycle targets recycling triggered by ultra-trace Hg2+ produce massive G-quadruplex fragments that bind haemin to yield G-quadruplex−haemin DNAzymes, which can efficiently catalyse ABTS-H2O2 system and convert ABTS2- to the coloured products. This ultra-sensitive colorimetric assay successfully achieved Hg2+ analysis in urine as low as 0.07 pmol/L with a linear calibration range from 0.1 pmol/L to 50 pmol/L (R2 = 0.992), and was amenable to quantification of Hg2+ in complex biological matrixes. This proposed single hairpin DNA-propelled signal amplification strategy displayed excellent sensitivity, remarkable specificity, high capacity of resisting disturbance, simplicity, low-cost, and was successfully applied for colorimetric detection of Hg2+ in urine samples with good recovery and accuracy.