<p>Development of ratiometric fluorescent probes for Cu²⁺ in aqueous solutions and biological systems remains the challenging task, given that Cu²⁺ commonly acts as an efficient fluorescence quencher. In this work, a novel dyad compound NI-SP bearing energy donor naphthalimide and energy acceptor styrylpyridine chromophore has been prepared using azide-alkyne click reaction. The photophysical properties of NI-SP and its coordination with Cu²⁺ have been investigated by the absorption and fluorescent spectroscopy. Upon addition of Cu²⁺ to a solution of NI-SP, the long wavelength emission peak of styrylpyridine (600 nm) was quenched, whereas the fluorescence of naphthalimide (450 nm) was enhanced due to a decrease in resonance energy transfer efficiency between the chromophores in the (NI-SP)·Cu²⁺ complex. The observed spectral changes enable ratiometric detection of Cu²⁺ by the registration of the ratio of fluorescence intensities <i>I</i>₄₅₀/<i>I</i>₆₀₀. The probe exhibited high selectivity toward Cu²⁺ in the tested conditions. The detection limit was determined at 120 nmol·L⁻¹, and the stability constant for (NI-SP)·Cu²⁺ was found to be 3.0 × 10⁶ L·mol⁻¹. Bioimaging experiments showed the NI-SP could penetrate human lung adenocarcinoma A549 cells, accumulate in mitochondria, and respond to the presence of Cu²⁺ via the changes in the fluorescence intensity of styrylpyridine fragment.</p>