The biomedical application of high-voltage nanosecond pulsed electric fields (nsPEFs) is an emerging interdisciplinary research field in recent years. Compared with microsecond and millisecond pulsed electric fields, high-voltage nsPEFs can not only lead to the polarization and dielectric breakdown of cell membrane structure, i.e. membrane electroporation, but can penetrate into the cell, triggering 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. This article first introduces the physical model of high-voltage nsPEFs and its bioelectrical effects on cellular organelles; then reviews and summarizes existing research on the interactions of high-voltage nsPEFs with cytoskeleton, mitochondria, endoplasmic reticulum, cell nucleus and other subcellular structures; the relationship between the impact 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 of high-voltage nsPEFs in biomedical research are condensed, followed by prospects of future research directions.