Background: The aim of this paper is to propose laws of trephine operation in order to obtain better microsurgical effects for keratoplasty. Methods: Based on a handheld trephine manipulator and a trephine robot integrated with the microforce sensor, the manual and robotic experiments are made taking porcine corneas as the test subjects. The effect of trephine operation parameters on the results reflected by biomechanical response and photomicrograph is discussed, and the parameters include linear velocity, rotating angle and angular velocity. Results: Using probability density functions, the distributions of the manual operation parameters show some randomness, and there is 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 cutting cornea more stably and produce a uniform cutting margin. Conclusions: Under different operation 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 operation parameters can be set in robotic trephination, and the surgeons can also be specially trained to achieve a better microsurgical result.