A probe calibration route simulation system and method is applied to a computer, and the computer comprises a storage device and a display device. The probe calibration route simulation method comprises the steps of: drawing a three-dimensional model of a standard ball and a probe according to surface node data and control point data of the standard ball and the probe; selecting measuring points on circle of every layer of the standard ball, calculating the coordinate of every measuring point, and putting the coordinate of every measuring point into a self-defining array; calculating the coordinate of moving points of the probe moving on the sphere and inserting the moving points between every two neighboring measuring points; simulating the moving route of the probe according to measuring points and the moving points and leading every point on the moving route to be subjected to probe collision detection; converting the moving route of the probe into a probe calibration program when the probe does not collide with the standard ball. According to the probe calibration route simulation system and method, by means of simulating the dynamic moving route of the probe on the standard ball sphere, the operation status of the probe can be timely and accurately reflected, and the probability of the collision between the probe and the standard ball can be eliminated.