Transport of protein and RNA cargoes between the nucleus and cytoplasm (nucleocytoplasmic transport) is vital for a variety of cellular functions. The studies of kinetics involved in such processes have been hindered by the lack of quantitative tools for measurement of the nuclear and cytosolic fractions of an intracellular protein at the single cell level for a cell population. In this report, we describe using a novel method, microfluidic electroporative flow cytometry, to study kinetics of nucleocytoplasmic transport of an important transcription factor NF-κB. With data collected from single cells, we quantitatively characterize the population-averaged kinetic parameters such as the rate constants and apparent activation barrier for NF-κB transport. Our data demonstrate that NF-κB nucleocytoplasmic transport fits first-order kinetics very well and is a fairly reversible process governed by equilibrium thermodynamics.