We present a method for the simultaneous measurement of 5 degree-of-freedom (DOF) spindle error motions in computer numerical control (CNC) machine tools and develop a measurement system. The measurement system uses polarization-maintaining fiber coupled with a dual-frequency laser as the light source. The axial error motion of the spindle is measured by heterodyne interferometry, and other 4DOF error motions are obtained by collimation measurement. Based on the measurement system, a comprehensive measurement model of spindle error motion was established. The influence of system error on the measurement accuracy of the spindle tilt error motion and radial error motion was analyzed. According to the measurement model, a corresponding data-processing method is proposed, which removes some systematic errors. A series of experiments was conducted to verify the feasibility and effectiveness of the proposed measurement system and the corresponding measurement model.