The longitudinal and reverse positioning accuracy and repeat positioning accuracy of the Y-axis in the CNC boring and milling machine were analyzed by using Renishaw laser interferometer. The measurement results were analyzed and discussed referring to the definition of the accuracy and the mathematical models of both the positive and reverse one-way positioning accuracy and reverse difference compensation of Y-axis were constructed. The positioning error and the reverse difference were compensated by the function of the screw and gap compensation table of 840D system. The test results show that the positive and reverse positioning accuracy has been greatly improved after compensation. There is a more obvious reduction in the backlash.