This study introduces an acoustothermal transfection method that enhances the permeability of both the cell membrane and nuclear envelope to achieve efficient and safe transfection of primary T cells and stem cells. The method leverages the combined effects of acoustic and thermal forces induced by surface acoustic waves (SAWs), which perforate the cell membrane and nuclear envelope, allowing for the delivery of large plasmids into the nuclei. The technique was validated through experiments on MCF-7 cells, mesenchymal stem cells (MSCs), and primary T cells, demonstrating high transfection efficiencies of up to 89.6 ± 1.2% for delivering dual plasmids. The method also showed high biocompatibility, maintaining cell viability and functionality, as evidenced by RNA sequencing and in vivo validation in a stroke model. The acoustothermal transfection technique addresses the fundamental trade-off between transfection efficiency and cell viability, making it a promising tool for personalized cell therapy and immunotherapy.This study introduces an acoustothermal transfection method that enhances the permeability of both the cell membrane and nuclear envelope to achieve efficient and safe transfection of primary T cells and stem cells. The method leverages the combined effects of acoustic and thermal forces induced by surface acoustic waves (SAWs), which perforate the cell membrane and nuclear envelope, allowing for the delivery of large plasmids into the nuclei. The technique was validated through experiments on MCF-7 cells, mesenchymal stem cells (MSCs), and primary T cells, demonstrating high transfection efficiencies of up to 89.6 ± 1.2% for delivering dual plasmids. The method also showed high biocompatibility, maintaining cell viability and functionality, as evidenced by RNA sequencing and in vivo validation in a stroke model. The acoustothermal transfection technique addresses the fundamental trade-off between transfection efficiency and cell viability, making it a promising tool for personalized cell therapy and immunotherapy.