We introduce color-shifting fluorophores that reversibly switch between a green and red fluorescent form through intramolecular spirocyclization. The equilibrium of the spirocyclization is environmentally sensitive and can be directly measured by determining the ratio of red to green fluorescence, thereby enabling the generation of ratiometric fluorescent probes and biosensors. Specifically, we developed a ratiometric biosensor for imaging calcium ions (Ca2+) in living cells, ratiometric probes for different proteins, and a bioassay for the quantification of nicotinamide adenine dinucleotide phosphate.