Defects generated by particles can be prevented carefully controlling the process, but defects generated in grain boundary have to be annealed to get rid of them. Organic materials have weakness for heat so annealing process has to be avoid. In this study, we developed Al2O3 nano-structure single gas barrier layer using Neutral Beam Assisted Sputtering (NBAS) process. The NBAS process can be continuously changing crystalline structure from amorphous phase to nano-crystal phase of various grain sizes within single inorganic thin film. These NBAS process effect can lead to formation of nano-structure barrier layer which effectively passivate and healing gas diffusion pathways between grain boundaries. As a result, we confirmed nano-structure of NBAS processed Al2O3 single gas barrier layer through the dielectric constant measurement, Ellipsometry, AFM, and TEM analysis. Finally, WVTR property of Al2O3 nano-structure single gas barrier layer is measured under 1ⅹ10-6gm-2day-1. The Al2O3 nano- structure single gas barrier layer is very suitable in the OLED application.