An ionic liquid (IL) coated capillary was prepared and investigated for DNA separation. The electroosmotic flow of the capillary was reversed between pH 4.5 and 9. Below 900 base pairs the larger DNA fragment suffered more retardation in the IL coated capillary due to the increasing charge density of the fragment with size. In the presence of 4% hydroxyethylcellulose, the ΦX174 DNA-Hae III digest fragments were baseline separated in both IL- and polyacrylamide-coated capillary except for the fragments of 271 and 281 base pairs; while the analysis time was shorter in the IL-coated capillary. Our experiments indicated that the IL-coated capillary could work stably in the run buffer for at least 96 h with no notable deterioration in performance.