Local Excitation Dominance Governs the Photophysics of Hemi‐Cyanine Dyes: Structural Modulation and Fluorescence Sensing of Butyrylcholinesterase

ABSTRACT Balancing red‐shifted emission and high luminescence efficiency remains a central challenge in developing near‐infrared (NIR) fluorophores. Conventional charge‐transfer (CT) designs can effectively red‐shift emission; however, excessive spatial separation of electron density often leads to fluorescence quenching and low brightness. A synergistic approach involving donor modification is reported, π‐bridge engineering, and ionic modulation to construct three series of D–π–A hemi‐cyanine dyes with Local Excitation (LE)‐dominated excited states. The optimized dyes exhibit NIR emissions up to 802 nm ( ε max × Φ = 1716 L mol −1 cm −1 ) and ultrahigh brightness reaching 10 799 L mol −1 cm −1 at 735 nm. Both theoretical calculations and photophysical analyses consistently indicate dominant local excitation (LE) character, evidenced by limited spatial separation between hole and electron distributions (Sr, D, t index) and negligible dependence of emission maxima on solvent polarity ( E T (30)). This LE‐preserving excited‐state design enables red‐shift extension without compromising fluorescence output. Moreover, the retained reactive hydroxyl site allows straightforward functionalization, leading to two “turn‐on” probes for selective detection of butyrylcholinesterase. This work establishes a structure–excited‐state–function design paradigm that achieves both spectral tunability and bio‐responsive functionality, providing a new direction for constructing bright, LE‐based NIR fluorophores for biological applications.