Efficient DNA electrotransfer into muscles can be achieved by combining two types of electronic pulses sequentially: short high-voltage (HV) pulse for the cell electropermeabilization and long low-voltage (LV) pulse for the DNA electrophoresis into cells. However, the voltages currently applied can still induce histological and functional damages to tissues. Pluronic L64 has been considered as a molecule possessing cell membrane-disturbing ability. For these reasons, we hope that L64 can be used as a substitute for the HV pulse in cell membrane permeabilization, and a safe LV pulse may still keep the ability to drive plasmid DNA across the permeabilized membrane. In this work, we optimized the electrotransfer parameters to establish a safe and efficient procedure using a clinically applied instrument, and found out that the critical condition for a successful combination of electrotransfer with L64 was that the injection of plasmid/L64 mixture should be applied 1 h before the electrotransfer. In addition, we revealed that the combined procedure could not efficiently transfer plasmid into solid tumor because the uncompressed plasmid may rapidly permeate the leaky tumor vessels and flow away. Altogether, the results demonstrate that the combined procedure has the potential for plasmid-based gene therapy through safe and efficient local gene delivery into skeletal muscles.