The introduction of phosphine oxide into a fluorescein scaffold has yielded phospha-fluorescein with bathochromically shifted spectra, reliable photostability and solubility. Moreover, ratiometric and turn-on fluorescence in the decaging process has ensured that the phospha-fluorescein is a unique scaffold for fluorescence bioimaging. Probe DiMe-PF-Gal without further structural decoration was designed for accurately monitoring β-galactosidase in vivo.