Herein a rapid and sensitive fluorometric bioanalysis platform for mercury(II) (Hg²⁺) detection was innovatively developed using ultrathin two-dimensional MXenes (Ti₃C₂) as fluorescence quencher and Hg²⁺-induced exonuclease III (Exo III)-assisted target recycling strategy for efficient signal amplification. Initially, fluorophore-labeled single-stranded DNA (FAM-labeled probe) can be easily adsorbed onto the surface of ultrathin Ti₃C₂ nanosheets by hydrogen bonding and metal chelating interaction, and the fluorescence signal emitted by the FAM-labeled probe is quenched strongly owing to the fluorescence resonance energy transfer between the FAM and ultrathin Ti₃C₂ nanosheets. Upon sensing the target Hg²⁺, the protruding DNA fragment at the 3′ end of hairpin will hybridize with primer (hairpin-Hg²⁺-primer), and then further digested by Exo III to produce a probe (nicker). The released target Hg²⁺ and primer continue to participate in the next recycling, resulting in more hairpin probes becoming nickers. The combination of a large number of nickers and FAM-probe resulted in a significant increase in the fluorescence signal of the system, which was attributed to the fact that the double helix DNA was more rigid and separated from the surface of the ultrathin Ti₃C₂ nanosheets. The obvious fluorescence signal change of the Ti₃C₂-based Exo III-assisted target recycling can be accurately monitored by fluorescence spectrometry, which is also proportional to the concentration of Hg²⁺. Under optimum operating conditions, the peak intensity (520 nm wavelength) of fluorescence increased with increasing Hg²⁺ within a wide dynamic working range from 0.05 nM to 50 nM (R² = 0.9913) with a limit of detection down to 42.5 pM. The proposed strategy uses ultrathin MXenes as a platform for binding nucleic acids, which contributes to its potential in nucleic acid hybridization-based biosensing and/or nucleic acid signal amplification bio-applications.