A rhodamine–pyridine conjugate, REDA-2PC, can selectively detect NCS⁻ in human embryonic kidney ​293 (HEK293) cells. DFT calculations suggest that this is due to non-covalent interactions (hydrogen bonding N–H⋯NCS⁻, π–π stacking) and long range electrostatic forces acting between the sulfur atom of the NCS⁻ anion and the NEt₂ unit of REDA-2PC. A visible light excitable probe allows fluorescence and naked eye detection of nanomolar NCS⁻, much lower than normal NCS⁻ levels in the human body. A “lock” and “key” sensing mechanism is established and compared with the corresponding fluorescein derivative as a model compound.