Inefficient and low-precision delivery of exogenous nucleic acids (ENA) severely limits gene therapy on ischemic stroke (IS). Two problems need to be urgently addressed to improve the efficacy of gene therapy; first, the blood brain barrier (BBB) should be open to promote the accumulation of ENA or genetic material carriers in the ischemic brain parenchyma, and second, the efficient delivery of ENA into the ischemic cells. Previous studies applied ultrasonic cavitation either for opening BBB or for inducing sonoporation to deliver genetic materials into cells. However, the effectiveness of the two-step ultrasonic cavitation to deliver ENA in the brain remains unclear, let alone the genetic materials to be controllably delivered into the ischemic brain parenchyma of the IS. This study systematically explored the BBB opening and ENA delivery by the two-step ultrasonic cavitation using artificial acoustic-cationic-polymeric-nanodroplets (ACPNs). The results demonstrated that the first focused ultrasound (FUS), set at parameters of 3.3 MPa, 20 Hz, 200 cycles and 5 s, stimulating intravascular ACPNs cavitation effectively opened BBB to allow nonactivated ACPN extravasation and accumulation into the ischemic brain parenchyma. Then, the extravascular ACPNs enhanced the second ultrasonic cavitation that noninvasively and efficiently controlled ENA delivery to the ischemic cells through sonoporation, particularly applying 3.3 MPa, 60 Hz, 200 cycles and 9 s to control FAM-eNA delivery, and 3.6 MPa, 20 Hz, 200 cycles and 7 s for pEGFP-C1 controlled delivery. Overall, the two-step ultrasonic cavitation represented a potential strategy for IS-targeted ENA controlled delivery.