Lysosomal pH plays an essential role in mediating various biological processes such as immunization, cell metabolism and enzyme activity. Herein, by utilizing the fluorescence resonance energy transfer (FRET) strategy, a two-photon (TP) ratiometric fluorescent probe (NpLys-pH) has been developed for tracking of lysosomal pH changes in living cells, tissues, and zebrafish. NpLys-pH was constructed by conjugating a pH turn-on TP fluorophore 1 (D-p-A-structured naphthalene derivative) with a pH turn-off naphthalimide fluorophore 2 via a non-conjugated linker. Meanwhile, NpLys-pH has two potential...