A method of forming a layout definition of a semiconductor device includes the following steps. Firstly, a plurality of first patterns is established to form a material layer over a substrate, with the first patterns being regularly arranged in a plurality of columns along a first direction to form an array arrangement. Next, a plurality of second patterns is established to surround the first patterns. Then, a third pattern is established to form a blocking layer on the material layer, with the third pattern being overlapped with a portion of the second patterns and with at least one of the second patterns being partially exposed from the third pattern. Finally, the first patterns are used to form a plurality of first openings in a stacked structure on the substrate to expose a portion of the substrate respectively.