To simplify the procedure of rotation axis positioning, a flexible and real-time calibration method is proposed. A plane calibration board with tessellated pattern is placed on the rotating stage, the calibration board was recorded at every point when the stage was rotated. Then the coordinates of these character points on the target are calculated by processing these pictures. At last, the axis equation of the working stage is acquired by linear fitting with the circle centers which were gotten by circle fitting with the character points. This method is proved by experiment, and the root-mean-square error of a measured object is 0.04 mm.