The invention belongs to the field of optical molecule imaging, in particular to an intraoperative tissue cherenkov fluorescence imaging system and an image processing method thereof. The problems that the material collection area of intraoperative frozen pathologic slices is small and the analysis time is long are solved. The system disclosed by the invention comprises a light source module, an information acquisition module, and a central control module, wherein the light source module is used for emitting white light irradiated intraoperative tissues; the information acquisition module is used for acquiring colored white light images, gray-scale white light images and cherenkov fluorescence images of the intraoperative tissues; the central control module is used for controlling the light source module to lighten and controlling the information acquisition module to acquire the images and receive the state information of the light source module and image information acquired by the information acquisition module; and the intraoperative tissue images are generated through a built-in image processing algorithm. According to the imaging system disclosed by the invention, spontaneoussignals are produced during imaging, a signal-to-background ratio is high, and an imaging process is high in specificity, high in sensitivity, high in resolution ratio of a shallow layer and high inimaging speed; and moreover, surgical margin testing of the intraoperative tissues can be quickly and effectively realized.