We report an easy and stable transfection technique using electrogene transfer with a nonviral Epstein-Barr (EB) virus-based vector. To achieve stable transfection of human breast cancer cells, we conducted electrogene transfer of an EB virus-based plasmid vector (reduced size of oriP) containing the enhanced green fluorescence protein (eGFP) gene. Because the EB virus-based vector exhibits high transfer efficiency and strong persistent transgene expression as a result of autonomous replication in human cells, and as Nucleofector electrogene transfer can achieve highly efficient gene transfection, this method is particularly suitable for generation of stably transfected cell lines.