The invention discloses an endogenous optical imaging method for automatically separating arteries and veins and aims at providing an effective endogenous optical imaging method for effectively and automatically separating arteries and veins of organisms. In the technical scheme, the endogenous optical imaging method comprises the following steps of: carrying out spectrum analysis to a collected image sequence by adopting narrow-band quasi monochromatic light for illumination, and then realizing the identification and automatic separation of the arteries and veins by utilizing the amplitude distribution features of low-frequency oscillating signals of the image sequence through the operations of frequency spectrum transformation, enhancement of a local self-adaption contrast, automatic threshold segmentation and the like. The endogenous optical imaging method can be used for automatically separating the arteries and the veins and highlighting and separating the vessel structures of many arterioles and veinlets, thereby avoiding the design complexity of the imaging device using light with various central wavelengths for illumination.