A method of forming an SOI semiconductor substrate and the SOI semiconductor substrate formed thereby, is provided. The method includes forming sequentially buried oxide, diffusion barrier and SOI layers on a semiconductor substrate. The diffusion barrier layer is formed by an insulating layer having a lower impurity diffusion coefficient as compared with the buried oxide layer. The diffusion barrier layer serves to prevent impurities implanted into the SOI layer from being diffused into the buried oxide layer or the semiconductor substrate.