A wafer on a robot blade may slip due to inertia sliding during the acceleration or deceleration process. This study presents the implementation and experimental verification of a novel real-time wafer positioning system to be used during the transfer process. A system-integration computer program involving a human-machine interface (HMI) was also developed, exhibiting the following functions: (a) moving direction judgment; (b) notch-passing judgment; (c) indicating the sensor by which the notch passes; and (d) computing the wafer center in real time. The position of the wafer center is calculated based on the time-sequence of the beam-breaking signals from two optical sensors, and the geometric relations among the sensing points of the robot blade and wafer. When using eight-inch wafers, the experimental results indicated the capabilities of the proposed positioning system under various conditions, including distinct parameters regarding the moving direction, wafer displacement and notch-passing sensors. The accuracy and precision (repeatability) of the measurement in various conditions were calculated and discussed. Furthermore, the experimental results demonstrate that, after combining the novel wafer positioning system and HMI program, the proposed method can be used to compute the position of the wafer center in real time in various conditions.