Single-cell electroporation is a powerful technique to understand cellular behavior with heterogeneity, which might be impossible based on bulk measurements of millions of cells together. In this study, a dielectric passivation layer was deposited on top of an indium-tin oxide micro-electrode-based transparent chip surface using a plasma enhanced chemical vapour deposition technique. We theoretically and experimentally investigated the key effects of the dielectric passivation layer on localized single-cell electroporation for different cancer cells, which were randomly distributed with a high density throughout the chip surface as a monolayer. The passivation layer not only prevented the conventional or bulk electroporation with bubble and ion generation, but also provide an intense electric field in-between electrode gap for localized single-cell electroporation with high cell viability. Thus, devices with dielectric passivation layers are potentially applicable for single-cell studies. © 2016 The Royal Society of Chemistry.