The physical fractionation of cytoplasmic versus nuclear components of cells is a key step for studying the subcellular localization of molecules. The application of an electric field is an emerging method for subcellular fractionation of proteins and nucleic acids from single cells. However, the multibiophysical process that involves electrical lysis of cytoplasmic membranes, electrophoresis, and diffusion of charged molecules remains unclear. Here we study RNA dynamics in single cells during the electrophoretic extraction via a microfluidic system that enables stringent fractionation of the subcellular components leveraging a focused electric field. We identified two distinct kinetics in the extraction of RNA molecules, which were respectively associated with soluble RNA and mitochondrial RNA. We show that the extraction kinetics of soluble RNA is dominated by electrophoresis over diffusion and has a time constant of 0.15 s. Interestingly, the extraction of mitochondrial RNA showed unexpected heterogeneity in the extraction with slower kinetics (3.8 s), while reproducibly resulting in the extraction of 98.9% ± 2% after 40 s. Together, we uncover that the microfluidic system uniquely offers length bias-free fractionation of RNA molecules for quantitative analysis of correlations among subcellular compartments by exploiting the homogeneous electrophoretic properties of RNA.