Superconducting quantum bits based on Al/AlOx/Al Josephson junction devices are among the most developed quantum bits at present. The microstructure of the device interface critically affects the electrical properties of Josephson junctions, which in turn severely affects the superconducting quantum bits. Further progress towards scalable superconducting qubits urgently needs to be guided by novel analysis mechanisms or methods to improve the performance of junctions. A direct experimental study of the atomic structure of the device is very challenging. Therefore, we simulated three-dimensional Al/α-Al2O3/Al Josephson junction devices via first-principles electronic structure and ballistic transport calculations to investigate the relationship between transport properties and the Al/Al2O3 stacking sequence. This work elucidates in detail the effects of the aluminum and alumina stacking sequence on the electron transport properties of the Al/Al2O3/Al system at the microscopic level by combining first-principles density functional theory and a non-equilibrium Green's function formalism. It is first revealed that the oxygen termination mode exhibits the least sensitivity to conductance changes in the Al/Al2O3 stacking sequence, offering useful theoretical guidance for increasing the yield of fixed-frequency multi-qubit quantum chips which require tight control on qubit frequency.