Electroporation has been one of the most popular non-viral technologies for cell transfection. However, conventional bulk electroporation (BEP) shows significant limitations in efficiency, cell viability and transfection uniformity. Recent advances in microscale-electroporation (MEP) resulted in improved cell viability. Further miniaturization of the electroporation system (i.e., nanoscale) has brought up many unique advantages, including negligible cell damage and dosage control capabilities with single-cell resolution, which has enabled more translational applications. In this review, we give an insight into the fundamental and technical aspects of micro- and nanoscale/nanochannel electroporation (NEP) and go over several examples of MEP/NEP-based cutting-edge research, including gene editing, adoptive immunotherapy, and cellular reprogramming. The challenges and opportunities of advanced electroporation technologies are also discussed.