Exogenous control of intracellular drug delivery improves efficacy by reducing nonspecific off-target toxicity. However, achieving precise on-demand dosage in deep tissues with minimal damage remains challenging. Here, we report an electric-pulse-driven nanopore-electroporation (nEP) system for localized intracellular delivery in deep tissues. Compared with conventional bulk electroporation, in vitro nEP achieved superior transfection efficiency (>60%) and high cell recovery (>95%) under nontoxic low voltage (40 V). Furthermore, in vivo nEP using a nanopore needle electrode with a side drug-releasing compartment optimized dosage, time, and location control of propidium iodide. In animal pilot studies, nEP exhibited two-fold higher transfection efficiency in thigh muscle while minimizing tissue damage (<20%) versus bulk electroporation. This tissue-penetrating nEP platform enables localized, safe, and effective intracellular delivery of diverse therapeutics into deep tissues.