<p>Trace element analysis of ancient human remains plays an important role in reconstructing dietary habits and living environments. However, conventional analytical methods have critical limitations such as destructive sampling, high costs, and inadequate sensitivity. To address these challenges, a reversible fluorescent probe with specific recognition for Cu²⁺ was fabricated and characterized in this study for cell detection and bioimaging. Compared with other cations in solution, the probe exhibited exceptional selectivity and sensitivity towards Cu²⁺. The interaction between the probe and Cu²⁺ accelerated the ring-opening process, resulting in a new emission band at 525 nm within a specific pH range. A strong linear relationship between the fluorescence intensity and Cu²⁺ concentration, ranging from 0.0 to 1.0 equivalents, with a detection limit of 4.52 μM, was observed. In addition, Job plots and infrared spectral analysis revealed that B probe complexed Cu²⁺ at a 1:1 ratio. Biological testing revealed that the probe has good biocompatibility and can be effectively used for Cu²⁺ cell imaging. Cu²⁺-specific reversible fluorescent probes can be designed and fabricated with good fluorescence response characteristics and combined with fluorescence spectroscopy technology to accurately quantify Cu²⁺ in complex ancient human samples.</p>