The efficient nonviral delivery of nucleic acids into the cytoplasm is needed to fully realize the potential of gene therapy. Although cationic lipids and nanoparticles have been widely used to improve the intracellular delivery of nucleic acids, they suffer from cytotoxicity and poor endosomal escape, thus limiting the transfection efficacy. Here, we developed a photothermal transfection platform for efficient and biosafe intracellular delivery of nucleic acids. Photothermal transfection was carried out by irradiation of cells co-treated with Lipofectamine-plasmid DNA complexes and PEGylated gold nanorods (GNRs) using an NIR laser for 30 min and subsequent incubation of the cells for 30 min without laser irradiation. Compared to conventional Lipofectamine-based transfection, our photothermal transfection platform significantly improved the transfection efficiency in difficult-to-transfect human primary cells including human dermal fibroblasts while maintaining the cell viability. The photothermal heating did not leave the GNRs inside the cell, thereby minimizing the cellular damage. Furthermore, the photothermal transfection platform showed superior genome editing abilities (both gene cleavage and insertion) in human dermal fibroblasts than conventional Lipofectamine-based transfection.