The aim of this paper is to propose laws of trephine operation based on a robot-assisted cutting cornea in order to obtain better microsurgical effects for keratoplasty. Using a trephine robot integrated with a microforce sensor and a handheld trephine manipulator, robotic and manual experiments were performed, with porcine corneas as the test subjects. The effect of trephine operational parameters on the results reflected by the biomechanical response is discussed, and the parameters include linear velocity, rotating angle, and angular velocity. Using probability density functions, the distributions of the manual operational parameters show some randomness, and there is a large fluctuation in the trephine force during the experiments. The biomechanical response shows regular trends in the robotic experiments even under different parameters, and compared to manual trephination, the robot may perform the operation of trephine cornea cutting more stably. Under different operational parameters, the cutting force shows different trends, and the optimal initial parameters that result in better trephine effects can be obtained based on the trends. Based on this derived law, the operational parameters can be set in robotic trephination, and surgeons can also be specially trained to achieve a better microsurgical result.