To obtain an ideal transfection efficiency of porcine fetal fibroblasts, fluorescence activated cell sorting (FACS) was used to optimize parameters for transfection of porcine fetal fibroblasts (PFFs) with ECM? 830, NEPA 21 and Nucleofector? 2b in different conditions such as electroporation parameters, plasmid dosages and topological structures. The results show that the optimum poring pulse parameter of NEPA 21 is voltage 200 V, continuous 3 ms, interval 50 ms, 3 times, voltage attenuation range of 10%; and the transfection efficiency of Nucleofector? 2b is highest under U-023 program. Under the optimum conditions, FACS analysis demonstrates that Nucleofector? 2b and ECM? 830 have the highest transfection efficiency when transfecting 10 μg supercoiled plasmids into PFFs, and 8 μg for NEPA 21. Supercoiled plasmids show higher transfection efficiencies than linearized plasmids. Moreover, Nucleofector? 2b has the highest transfection efficiency among the three electroporation instruments. This study paves the way to generate transgenic or gene editing pigs with high efficiency.