A simple, sequential DNA pre-concentration and separation method by using a micro-CE chip integrated with a normally closed valve, which is activated by pneumatic suction, has been developed. The CE chip is fabricated using PDMS. A surface treatment technique for coating a polymer bilayer with an anionic charge is applied to modify the surface of the microchannel. A normally closed valve with anionic surface charges forms a nanoscale channel that only allows the passage of electric current but traps the DNA samples so that they are pre-concentrated. After the pre-concentration step, a pneumatic suction force is applied to the normally closed valve. This presses down the valve membrane, which reconnects the channels. The DNA samples are then moved into a separation channel for further separation and analysis. Successful DNA pre-concentration and separation has been achieved. Fluorescent intensity at the pre-concentration area is increased by approximately 3570 times within 1.9 min of operation. The signals from the separation of phiX174 DNA/HaeIII markers are enhanced approximately 41 times after 100 s of pre-concentration time, as compared with the results using a traditional cross-shaped micro-CE chip. These results clearly demonstrate that successful DNA pre-concentration for signal enhancement and separation analysis can be performed by using this new micro-CE chip.