In recent years, more studies focused on single cell electroporation. However, not only the position cannot be confined in the specific region of the cells, but also the employed electrodes are not transparent, causing difficulty on optical observation. Here, we demonstrated localized single cell membrane electroporation (LSCMEP), which an efficient to deliver drugs into single cell by selective and localized way from millions of cells. The ITO microelectrodes fabricated by wet etching as well as FIB technique, which successfully deployed 1μm gap between two microelectrodes. Due to submicron level gap, electric field can more intense in a narrow region. The diffusion mechanism with PI dye intensity variation has been analyzed by image pro plus software. In our approach, we have successfully achieved 0.99 μm electroporation regions on the cell membrane to inject PI dye into the cell with high cell viability by trypan blue test. We demonstrated the cell self-recover process from the injected PI dye intensity variation. Our localized single cell membrane electroporation technique, not only generates well-controlled nano-pore allowing rapid recovery of cell membrane, but also provides a clear optical path potentially monitoring drug/DNA delivered into single cell level even in single molecule level.