The presently disclosed subject matter provides thiol-reactive, environmentally sensitive fluorescent dyes, or fluorophores, which have an emission wavelength in the visible spectral region. When conjugated with a binding protein, the fluorophores exhibit a ratiometric response to one or more ligands or target analytes. The presently disclosed fluorophore-binding protein conjugates can be used to detect the presence of or amount of physiologically-important metabolites, such as glucose, fatty acids, and lactate, in biological samples.