By selecting laser power, laser pulse frequency and scanning speed as optimization variables, we establish a multi-objective optimization model of laser closure process parameters in vitro skin tissue. Based on MATLAB software, we use second generation non-dominant sequencing genetic algorithm (NSGA-II) to find the Pareto optimal solution set, obtain the optimal process parameters, and then analyze the response sensitivity of optimization objectives to the variation of process parameters. Under the optimized process parameters, the tensile strength of the incision is tested and the microstructure is analyzed. The results show that the incision tensile strength has high sensitivity to the laser process parameters, and the laser power has significant effect on the incision tensile strength and the tissue peak temperature. The proposed optimized process can achieve the in vitro skin tissue closure in full-thickness. In the case of tissue peak temperature decreasing, the tensile strength of in vitro skin tissue incision is 5.6% higher than that of single-objective optimization.