Nature endows us with a unique toolbox of highly specific enzymes, while their detection is of great importance in biological processes. The label-free assay based on DNA-templated CuNPs is widely accepted for enzyme assay owning to its simple procedure, fast kinetic, high quantum yield, and large Stokes shift. A challenge in the application of them is the low fluorescent signal stability of DNA-templated CuNPs, whose signal sharply decreases in a few minutes after formation. In this work, a long-term stable nuclease assay is proposed by utilizing the elemental mass spectrometry detection of CuNPs. The high sensitivity was also realized, thanks to a great number of copper isotopes (63Cu and 65Cu) intrinsically incorporated in CuNPs. The experimental conditions, including the length of polyT ssDNA template, the concentration of polyT template, the concentration of Cu2+, the sodium ascorbate concentration, the copper reduction reaction time, and the Exonuclease I (Exo I) digestion reaction time, were investigated in detail. The dynamic range of the Exo I concentration from 0.1 U/mL to 20 U/μL was obtained using inductive coupled plasma mass spectrometry (ICPMS) 63Cu signal, with a detection limit (3σ) of 0.029 U/mL. The ICPMS 63Cu signal remained unchanged for at least 18 days. The spiked-recovery assay in RPMI 1640 cell medium also demonstrated the reliability of the proposed nuclease assay.