In this paper, the main sources of fixture errors were analyzed, and an effective fixture positioning process for five-axis CNC machine tools was proposed, which improved positioning accuracy of using the traditional fixture positioning method. Considering the settings of the tilted work plane (TWP) compensation, the obtained positions of fixture locators, and the homogeneous transformation matrix, the analyzing results presented in this paper can be used to calculate the position of fixture locators and then to predict the position and orientation of the workpiece placed on the fixture. Therefore, the program coordinate frame can be shifted and oriented to the reference coordinate, and can reduce the fixture positioning errors.