Naphthalimide-based fluorescent probes (NAN0-N₃ and NAN6-N₃) were developed with dual locked fluorescence. Here, ≥1.9 × 10⁻² mM of H₂S and ≥2.2 × 10⁻² μg mL⁻¹ of DNA could unlock a highly sensitive off–on fluorescence response through synergistic changes of the molecular structure and conformation. As such, the probes could monitor DNA damage induced by the overexpression of H₂S, and were able to evaluate the degree of apoptosis of living cells mediated by H₂S-induced mtDNA or nDNA damage.