Abstract: Glass is a popular material for microfluidic chip,but its fabrication process includes pattern transferring and window-opening on sacrificial metal layer by photolithography is time consuming and high cost.Here a simple and rapid method for window-opening was developed.Cr/Au sacrificial film was developed by electrochemical micromachining using a PET through-mask ablated with CO₂ laser system.Window-opening could be finished within 10 seconds,and its quality depends on the thickness of the PET mask,component of electrolyte,the pressure and electric voltage applied.The width and depth of the fabricated microchannel are 145μm and 25μm respectively,relative standard deviation is 3.72%.Electrophoretic separation of amino acid mixture was carried out successfully using the fabricated glass chip.And gold microelectrode array with 100μm width and 100μm interval was fabricated by this method too.