We review the on-line concentration and separation of DNA and proteins in the presence of electroosmotic flow (EOF) in capillary electrophoresis (CE). The effects of concentrations of Tris-borate (TB) and poly (ethylene oxide) (PEO), pH and sample matrix on resolution and concentration factor are discussed. We also review stepwise techniques for DNA separations using different PEO solutions. Moreover, the techniques allowing on-line concentration and separation of up to 2.1-μL proteins or 5-μL DNA samples and their applications to the analysis of PCR products and urine samples have been discussed.