A method of forming an isolation layer includes forming mask pattern structure on a substrate to partially expose the substrate, etching the substrate using the mask pattern as an etching mask to form a trench, forming an impurity diffusion region at an inner face of the trench, and filling the trench with the isolation layer. A method of manufacturing an image device includes the method of forming an isolation layer, and at least additionally forming unit pixels including a photo diode and transistors on an active region defined by the isolation layer.