Quantitative determination of toxic cyanide anions (CN⁻) in biological samples is an important subject. We synthesized a naphthalimide–coumarin conjugate with a simple imine linker (1), which behaves as a fluorescent receptor for ratiometric quantification of CN⁻. It exhibits green naphthalimide fluorescence at 533 nm, originating from the excited-state intramolecular charge transfer (ESICT) from coumarin to naphthalimide fluorophores. Selective CN⁻ addition to the imine linker affords an α-amino nitrile product. This suppresses ESICT and creates coumarin fluorescence at 444 nm and naphthalimide fluorescence at 533 nm originating from direct photoexcitation of the respective fluorophores. The emission intensity at 533 nm is maintained upon CN⁻ addition and can be used as an internal standard for analysis. This ratiometric response enables self-calibrating CN⁻ quantification based on the intensities of two emissions in solution and in cells. This facilitates rapid (within 3 min), selective, and sensitive ratiometric detection of very low levels of CN⁻ (>1.8 μM).