A method of forming an isolation layer of a semiconductor device includes forming first trenches in an isolation region of a semiconductor substrate. Sidewalls and a bottom surface of each of the first trenches are oxidized by a radical oxidization process to form a first oxide layer. An oxidization-prevention spacer is formed on the sidewalls of each of the first trenches. Second trenches are formed in the isolation region below the corresponding first trenches, wherein each second trench is narrower and deeper than the corresponding first trench. The second trenches are filled with a second oxide layer. The first trenches are filled with an insulating layer.