A device isolation structure of semiconductor device includes a semiconductor substrate having a cell region, a low voltage region and a high voltage region defined therein. A cell trench isolation region is disposed in the cell region. A low voltage trench isolation region is disposed in the low voltage region and extends deeper into the substrate than the cell trench isolation region. A first high voltage trench isolation region is disposed in the high voltage region and extends deeper into the substrate than the low voltage trench isolation region. A second high voltage trench isolation region is disposed in the high voltage region and extends deeper into the substrate than the low voltage trench isolation region but shallower than the first high voltage trench isolation region.