Fluorescent probes are essential for the study of biological systems. Using two-photon excited fluorescence microscopy, real-time three-dimensional(3D) dynamic observation and monitoring of biologically active compounds and biological function of object composition can be intuitively and conveniently realized. In recent ten years, the developed two-photon excited fluorescence probes, compared with the one-photon fluorescence probes, have significant advantages, such as high resolution, high clarity, high sensitivity, no photobleaching and no photodamage, fixed target excitation, high transverse and longitudinal resolutions, small absorption coefficient of light in tissue, lower tissue auto. fluorescence, etc. The structures, characteristics and applications in biological imaging of two-photon fluorescence probes for cations, anions, SO2, pH, nucleic acid, cysteine, reactive oxygen species, phosphate, CO, alph-cells, CYP1A enzyme, and polarity, viscosity and temperature in recent seven years are reviewed. Two-photon fluorescent probes have been widely used in clinical diagnosis, disease monitoring and drug screening, which has promoted the development of biochemistry, medicine and life sciences.