Introduction of foreign macromolecules into single living cells with high transfection efficiency, high cell viability and high throughput are challenging task in cell biology and therapeutic research. Here we developed a 3D massively-parallel high throughput single-cell electroporation (MSEP) chip, which uniformly transfect single mammalian cells in parallel fashion with low applied voltages (3V~15V) and low signal (10KHz~10MHz) and achieved high delivery efficiency with high cell viability. This device have approximate 5000 holes (each hole diameter ~ 15µm) in parallel fashion on silicon substrate and electric field confined on top of each holes. When cells with cargo passes through each holes, electric field highly intense in each cells, resulting cell membrane deformation and create transient membrane pores to deliver exogenous cargo into cells. Our platform have ability to deliver molecules in 10 million cells/min by using 1 cm2 chip size. Using this MSEP device, we successfully deliver different sizes of dextran proteins, GFP plasmids into both adherent (HeLa) and non-adherent (THP-1) cells using different flow rates. This uniform single cell transfection with high efficiency and cell viability might be potentially application for single cell therapy, diagnostics and regenerative medicine purpose. Copyright © (2018) by Chemical and Biological Microsystems Society. All rights reserved.