A moisture barrier layer with high density and uniformity is essential for increasing the lifespan of optoelectronic devices such as flexible organic solar cells, LEDs, and photodetectors. In this study, various surface pre-treatments (O₂ plasma, UVO treatment, Al seed layer, and thermal annealing process) were performed on a polyethylene naphthalate (PEN) film to form a functional group of chemical adsorption with the atomic layer deposition (ALD) precursor. We investigated the effect of the surface pre-treatment of a PEN substrate for a deposition of Al₂O₃ thin film in terms of the deposition uniformity and water vapor transmittance rate (WVTR). For example, the root mean square roughness of the bare PEN film/Al₂O₃ was R_RMS=3.26 nm, whereas the O₂ plasma treated PEN film/Al₂O₃ had R_RMS=0.99 nm because of the presence of a functional group. As a result, WVTR of bare PEN film/Al₂O₃ was 0.83 g/m²/day, whereas that of O₂ plasma treated PEN film/Al₂O₃ decreased to 0.38 g/m²/day.