In order to improve the on-line inspection accuracy of Computer Numerical Control (CNC) machine tools, the influence of the positioning errors of each axis of machine tools on the on-line inspection accuracy of CNC machine tools is studied. Error compensation of CNC machine tool was carried out through experimental research. Firstly, a laser interferometer was used to measure the positioning errors of each axis on CNC machine tool. Then the positioning errors of each axis were compensated sequentially, and the algorithm of a cubic spline curve was written by taking Visual C++60 as the development tool. The measured data points were fitted into a curve to achieve the error effect that could predict any point of the machine tool, and finally the standard block test was performed. The results show that the error compensation effect is obvious, and the compensation software can be used to compensate the machine tool at any point.