Gene transfection is an important technology for various biological applications. The exogenous DNA is commonly delivered into cells by using a strong electrical field to form transient pores in cellular membranes. However, the high voltage required in this electroporation process may cause cell damage. In this study, a dielectrophoretically-assisted electroporation was developed by using light-activated virtual microelectrodes in a new microfluidic platform. The DNA electrotransfection used a low applied voltage and an alternating current to enable electroporation and transfection. Single or triple fluorescence-carrying plasmids were effectively transfected into various types of mammalian cells, and the fluorescent proteins were successfully expressed in live transfected cells. Moreover, the multi-triangle optical pattern that was projected onto a photoconductive layer to generate localized non-uniform virtual electric fields was found to have high transfection efficiency. The developed dielectrophoretically-assisted electroporation platform may provide a simpler system for gene transfection and could be widely applied in many biotechnological fields.