A non-volatile memory device and its manufacturing method are provided. The method includes the following steps. A plurality of isolation structures are formed in a substrate. A first polycrystalline silicon layer is formed in the substrate and between two adjacent isolation structures. A first implantation process is performed to implant a first dopant into the first polycrystalline silicon layer and the isolation structures. A portion of each of the isolation structures is partially removed, and the remaining portion of each of the isolation structures has a substantially flat top surface. An annealing process is performed after partially removing the isolation structures to uniformly diffuse the first dopant in the first polycrystalline silicon layer. A dielectric layer is formed on the first polycrystalline silicon layer, and a second polycrystalline silicon layer is formed on the dielectric layer.