A method of manufacturing an isolation structure suitable for a non-volatile memory is provided. A substrate is provided. A dielectric layer, a conductive layer, and a hard mask layer are sequentially formed on the substrate. The hard mask layer and the conductive layer are patterned to form a first trench which exposes the dielectric layer. A first liner is formed on the substrate. The first liner and the dielectric layer that are exposed by the first trench are removed to expose the substrate. A spacer is formed on sidewalls of the conductive layer and the hard mask layer. The substrate is partly removed to form in a second trench with use of the conductive layer and the hard mask layer with the spacer as a mask. An isolation layer is formed in the second trench. The distance between the conductive layers is greater than the width of the second trench.