The biaxial straightness of a numerical control machine tool is measured by a self-developed 6-degree-of-freedom (6DOF) motion error simultaneous measurement system based on single fiber-coupled dual-frequency laser. Through the linear fitting of the two-axis straightness error, the angle between the two fitting axes is obtained, and the vertical measurement of the two axes is indirectly realized. The corresponding error model is established by using ray vector tracing method, and the influence of installation and processing error of the pentagonal prism on the measurement error are analyzed. The experimental results show that our system has high accuracy. Compared with commercial measuring instruments, the difference between the measured results is within 1″.