The optical path calibration process using a laser interferometer is difficult. It needs to be adjusted repeatedly, which takes a long time. Also, it requires the skill level and experience of the operator, leading to a significant reduction in measuring efficiency.The optical path calibration process of the Y-axis of a three-axis machine tool was analyzed by Renishaw XL-80 laser interferometer.The homogeneous coordinate transformation error model of the measured axis was established.By means of the fixed distance from the center of the light target to the edge of the light target as the light spot moves, the laser transmitter yaw and pitch angle, which were independent of the Y-axis position of the measured axis, were solved. The laser and mirror position offset errors and the distance that the spot changed between coordinate systems were indirectly obtained. With the established error model, the yaw error of the beam splitter and the mirror around their respective vertical base axes that could not be directly measured were obtained. The components were adjusted to complete the optical path calibration process quickly and accurately.