Provided is a method of manufacturing a memory device including following steps. A substrate including an active region and a periphery region. A stack layer is formed on the substrate. A first trench is formed in the substrate and the stack layer in the active region. A first isolation structure is formed in the first trench. An ion implantation process is performed to form a doped first isolation structure. A first wet etching process is performed to remove a portion of the doped first isolation structure, so that a first recess is formed on the doped first isolation structure. A protection layer is formed on the substrate to at least cover sidewalls of the first recess. A second wet etching process is performed to remove the protection layer and another portion of the doped first isolation structure and deepen the first recess. A SICONI etching process is performed.