A simple negative pressure pinched sample injection method was presented. This method combined diaphragm micropumps and a single voltage supply to generate controllable well-defined sample plug, and led to effective electrophoresis separation. The pinched plug was found to be favorable for obtaining representative and reproducible results that the RSD of the migration time and peak height of sodium fluorescein were 0.5 and 2.1%, respectively (n = 25). The established method had been applied in separation of amino acid samples. This method has the advantages of well-defined plug, free sample bias effect, high reproducibility and convenience of controlling the negative pressure by the integrated pumps on the microchip. In addition, the single high voltage supply and the world-to-chip interface simplified the instrumentation, which is of benefit to the minimization and automation. These advantages demonstrate the potential of this method for a wide range of applications.