The invention discloses a real-time pupil tracking system and a fatigue state monitoring method. The system comprises a first infrared light source which generates 900 nm waveband infrared light; a second infrared light source which generates 850 nm waveband infrared light; a first video acquisition module which acquires an image reflected by the first infrared light source on a face; a second video acquisition module which acquires an image reflected by the second infrared light source on the face; and a video processing module which performs differential comparison on the images reflected by the first infrared light source and the second infrared light source and locates positions of human eyes. According to the invention, the positions of the human eyes are rapidly positioned through differences of eye retinas for infrared light reflectivity, and real-time tracking and analysis are realized through a whole-hardware image processing mode.