Abstract: The measurement uncertainty of the average grain size was evaluated for equiaxed grains by means of optical microscope eyepiece and manual counting method with analysis software. The main sources affecting measurement uncertainty were analyzed. The results show that the average intercept measured by eyepiece is 35.7 μm, U=2.1 μm, k=2. The average intercept measured by analysis software is 35.5 μm, U=0.4 μm, k=2. The main source of the average grain size measurement uncertainty with eyepiece is the test line length due to the microscope magnification deviation. The main source of the average grain size measurement uncertainty with analysis software is the deviation of intersection points between the grain boundary and the test line. Under the same conditions, the manual measurement average grain size with analysis software can reduce the uncertainty range of the results in the same probability. It can improve the accuracy compared with direct measurement using eyepiece. There is little difference between straight line intersection point method and circular intersection point method for the equiaxed grains in the average grain size uncertainty evaluation.