Despite the significant research in electroporation, high electric field was applied to the whole cells resulted in permeabilizing the membrane of millions of cells without reversibility . To deliver biomolecules through the specific region of the cell membrane with high cell viability and high transfection rate is important for various biological and therapeutic applications.This report presents a new type localized single cell membrane electroporation (LSCMEP), at specific region of the single cell withthe application of 800 μs electric pulse. The ITO nano-electrodes with 100nm thickness and 500 nm gap between two electrodes can generate an intense electric field to track biomolecules inside HeLa cell in our studies. This small gap between two nano-electrodes can neglect thermal effect on cell membrane and permit reversible electroporation with high cell viability (90%) and minimum effected electroporation region (0.48 μm). Our approach successfully delivers biomolecules through a specific region of single cell with high transfection rate (82%) and high cell viability. This process, not only generates well-controlled nano-pores allowing rapid recovery of cell membrane, but also it provides a clear optical path potentially tracking of drugs to deliver inside single cell. © 2013 IEEE.