Two-phase electrophoresis, coupling traditional solvent extraction with electrophoresis, is a novel separation technique. Being bio-compatible, aqueous two-phase electrophoresis provides a successful method for separating mixtures of biomolecules. In this work, the separation of amino acids by aqueous two-phase electrophoresis was examined with dextran-polyethylene glycol-water as a working system. The influences of separation time, field strength, initial solute concentration and two-phase contact area were studied in intermittent separation equipment. The experiment results show that aqueous two-phase electrophoresis is an effective separation technique for amino acids. With time, field strength, and contact area increasing, mass fluxes across the interface are increased quickly.