We present a prototype nanofluidic device, developed for the continuous-flow dielectrophoretic (DEP) fractionation, purification, and quality control of sample suspensions for gene vaccine production. The device consists of a cross injector, two operation regions, and separate outlets where the analytes are collected. In each DEP operation region, an inhomogeneous electric field is generated at a channel spanning insulating ridge. The samples are driven by ac and dc voltages that generate a dielectrophoretic potential at the ridge as well as (linear) electrokinetics. Since the DEP potential differs at the two ridges, probes of three and more species can be iteratively fully fractionated. We demonstrate the fast and efficient separation of parental plasmid, miniplasmid, and minicircle DNA, where the latter is applicable as a gene vaccine. Since the present technique is virtually label-free, it offers a fast purification and in-process quality control with low consumption, in parallel, for the production of gene vaccines.