The invention discloses a rotation matrix calibration method based on transverse moving motion in a TCP coordinate system. The method comprises the steps that firstly, a standard ball is set, a robotis controlled to carry a scanner to do transverse moving motion in three axis directions of the TCP coordinate system in the TCP coordinate system, and in the motion process, the standard ball is scanned; point cloud data of the standard at the different positions in the motion and coordinates of the ball center in the scanner coordinate system are obtained; and calculation is carried out to obtain three orthogonal unit transverse moving vectors of the ball center in the TCP coordinate system and the scanner coordinate system, and according to the corresponding transverse moving vectors, calibration of a rotation matrix can be finished. The automatic calibration flow is adopted, calibration of the rotation matrix can be finished through three times of transverse moving in the three coordinate axis directions in the TCP coordinate system, characteristic points, a calibration plate and the like do not need to be set, operation is easy, the calibration efficiency is high, the clamping error can be avoided, the calibration precision is high, and the method is suitable for measuring and machining integration of complex part robots in fields of machines, cars and the like.