The biomedical application of high-voltage nanosecond pulsed electric fields (nsPEFs) has become an emerging interdisciplinary research field in recent years. Compared with microsecond and millisecond pulsed electric fields, high-voltage nsPEFs can not only lead the cell membrane structure to polarize and dielectric break down the cell membrane structure, i.e. membrane electroporation, but also penetrate into the cell, triggering off organelle bioelectrical effects such as cytoskeleton depolymerization, intracellular calcium ion release, and mitochondrial membrane potential dissipation. Extensive attention has been attracted from related academic communities. In this article, the following aspects are involved. First, the physical model of high-voltage nsPEFs and its bioelectrical effects on cellular organelles are introduced. Then, the existing researches of the interactions of high-voltage nsPEFs with cytoskeleton, mitochondria, endoplasmic reticulum, cell nucleus and other subcellular structure are reviewed and summarized; the relationship between the influence on cellular organelles by high-voltage nsPEFs and the biological effects such as cell death and intercellular communication is highlighted. Finally, the key technical challenges to high-voltage nsPEFs in biomedical research are condensed, followed by the prospects of future research directions.