This article proposed calibration results of a real-time wafer positioning system using machine vision. We developed a novel wafer positioning system which comprises two line-shaped beams spreading toward a scattering surface on the way of wafer transfer. A CCD (charge coupled device) camera mounted above the scattering surface takes images, and these images are processed and analyzed to compute the actual position of the wafer center on the blade during the transfer process. A calibration process and linear fitting of the wafer center are proposed to compensate the system errors induced from manufacturing and assembly. Experimental results illustrate that the precision and accuracy of this wafer positioning system have been both improved after calibrating the system errors.