Objective To investigate the factors affecting the success rate and cell survival rate of electroporation-mediated transfection of human primary fibroblasts by optimizing the electroporation parameters, so as to screen the optimal conditions for electroporation-mediated transfection of human primary fibroblasts. Methods Plasmid pEGFP-N1 containing enhanced green fluorescent protein (EGFP) gene was transfected into human primary fibroblasts with electroporation by changing the conditions such as pulse voltage, pulse number and plasmid concentration. Cell mortality rate was detected by trypan blue staining. Transfection efficiency was measured by flow cytometry at 24 h after transfection. Results The electroporation-mediated transfection effect of human primary fibroblasts was optimal under the following condition:low voltage mode, pulse voltage of 320 V, plasmid concentration of 20 μg/mL and single pulse, with the cell transfection efficiency being (42.53±0.63)% and the cell survival rate being (74.30±3.01)%. Conclusion The electroporation-mediated transfection efficiency of primary human fibroblasts can be improved by optimizing the transfection conditions, which might lay a foundation for subsequent function research of gene.