Multiphoton microscopy (MPM) technology offers non-destructive, non-labeled imaging of tissue by detecting optical signals such as two-photon excitation fluorescence and second harmonic produced by the interaction of femtosecond laser with internal components of biological tissue. MPM has the advantages of tissue microstructure sensitivity, high spatial resolution imaging, low killing of biological tissues, deep imaging depth, and the ability to obtain the biochemical information of the tissue. It has great potential in disease diagnosis. The review mainly focuses on the principle of MPM and its applications in disease diagnosis of gastrointestinal cancer, skin diseases and corneal diseases, and looks forward to the development of MPM.