Gene transfection is an important technology in biological applications. Electroporation is one of gene transfection methods that deliver extracellular genetic materials into cells by using a high electrical field to forming pores on cell surface. However, the high voltage provided by an electroporator may impair cells to survive and cause low efficiency of yield rate. In this study, a new platform was developed for gene transfection under a lower applied voltage by utilizing an optical induced non-uniform electric field. To obtain higher transfection efficiency, a multi-spot optical image was projected to the gene transfection chip, resulting in localized non-uniform electric fields generated from these optical patterns. The experimental results in this study showed that one or more plasmid carried fluorescence genes could be effectively transfected into mammalian cells and the cells successfully expressed foreign proteins by using the developed optical-induced gene transfection platform. Moreover, the transfection efficiency of optical-induced gene transfection platform was significantly higher than the traditional electroporation technology. This developed platform may provide a simpler and more efficient tool for gene transfection.