The formation of diffusion barrier on the Ni-based superalloy substrate by low-pressure pre-oxidation is studied in this paper. The pre-oxidation was carried out by heating the substrate under a low-oxygen partial pressure with electron beam of the electron beam physical vapor deposition (EB-PVD) facility. The effect of diffusion barrier was investigated by both isothermal and cyclic oxidations at 1373 K, and the oxidation-kinetic curves were then obtained. The diffusion barrier mainly contains NiO,
,θ-Al
2O
3 and Cr
2O
3, and has a thickness of 185–225 nm. The investigation indicates that the barrier could partially inhibit the interdiffusion of elements of the superalloy substrate and then improve the oxidation-resistance performance of thermal barrier coatings (TBCs) to some extent. However, the difference of thermal cycling lifetime between TBCs samples with and without diffusion barrier is not extremely visible.