Metal nanoclusters have received considerable interest due to their unique properties and potential applications in numerous fields. Particularly, newly emerging Cu nanoclusters offer excellent potential as functional biological probes. In this work, we for the first time report that the fluorescence of DNA-hosted Cu nanoclusters is very sensitive to base type located in the major groove. This intriguing finding provides a sensitive fluorimetric diagnostic of the mismatch type in a specific DNA sequence, which is difficult to achieve by traditional methods. Furthermore, the research results have shed some light on the luminescent mechanism of Cu nanoclusters. Owing to its high specificity and easy operation without rigorously controlled temperature and arduous probe DNA design, it is expected that the proposed procedure can provide a tool for early diagnosis and risk assessment of malignancy.