For measuring the parallelism between the Z guide and the rotating axis of spindle on the machine tool, a new detection method which combines a machine tool probe based on laser displacement sensor with the approximation hole center positioning method is proposed. On the rule of keeping the rotating axis invariant, the same center of a ring gauge which lies on different points of the rotating axis is located twice and the obtained coordinate values can be used to calculate the parallelism. The detection method can eliminate some of the drawbacks involved in the measurement methods with the physical mandrel, meanwhile, they can achieve measurement as the spindle is rotating in high speed. The experimental results show that this method can complete parallelism measurement of the guide and the rotating axis of spindle, so it is expected to be a new means for the calibration of machine tools.