Mitochondria plays a key role in energy metabolism of cells,and the small changes of internal environment in mitochondria will affect the normal cell life activities.In the meanwhile,many active small molecules in mitochondria also play a key role in many physiological process.Therefore,visual monitoring of mitochondrial itself and its changes of the internal micro environment are of great importance for the research of biological phenomenon，disease diagnosis and treatment.Fluorescence method has been extensively used due to its advantages of simple operation,high sensitivity and selectivity,real time detection and little damage to organisms.The two photon fluorescent probe technique has shown a much broader application prospect in the field of life sciences compared to the one-photon fluorescent probe technique as it has many advantages,such as absorbing light of longer wavelength with lower energy but emitting shorter wavelength，high three-dimensional spatial selectivity，large penetration depth，avoidance of photodamage and photobleaching and lower tissue auto-fluorescence.The basic principle of organic two photon absorption and the research status of organic two-photon fluorescent probes for active small molecules in mitochondria were introduced,as well as the trends of organic fluorescent probes for active small molecules in mitochondria in the future were prospected.