Dynamics of oxygen vacancies play an important role in optimizing the properties of oxide materials, including oxygen permeability, oxygen ionic conductivity, catalytic activity, etc. A layered double perovskite oxide has attracted much attention due to its high oxygen transport rates. The layered double perovskite has a composition formula of AA`B2O5+with a stacking sequence of [A`O]-[BO2]-[AO]-[BO2] such as LnBaCo2O5+ (Ln = Pr, Nd, Sm, and Gd). The distribution of oxygen vacancies has been verified by the analysis of the oxygen site occupancy in NPD and intensity profile in TEM, but direct imaging of oxygen vacancies has remained a challenge. Here, we report direct atomic-scale visualization of the oxygen vacancy in the layered double perovskite oxide using an aberration corrected scanning TEM (STEM). Furthermore, order-disorder transition of oxygen vacancy is observed as temperature increases from room temperature to SOFCs operating temperature in an in-situ heating TEM.