It is one of the effective strategies to develop intermediate-temperature solid oxide fuel cells (IT-SOFCs) by looking for alternative electrolyte materials that can maintain or even improve current cell efficiencies while reducing operating temperature. In this study, by rationally introducing pentavalent Nb on the B-site of La0.6Sr0.4FeO3-δ (LSFO), a serial of novel perovskite La0.6Sr0.4Fe1-xNbxO3-δ (LSFNbx, x = 0.1, 0.2) with mixed electron-ion conduction is successfully prepared. The electrochemical mechanism and performance of the symmetrical IT-SOFCs with a single LSFNbx perovskite oxide as electrolyte are investigated. With the Nb doping, the structure stability of the LSFO is improved and the electronic conductivity decreases. La0.6Sr0.4Fe0.8Nb0.2O3-δ (LSFNb20) is the most promising electrolyte of the three candidates as it has the best performance (735 mW cm-2 at 550 °C in a dry H2/Air atmosphere) and no short-circuiting problem. The applied voltage-response current curves demonstrate that the interface between the Ni-Ni0.8Co0.15Al0.05LiO2-δ anode and LSFNb20 electrolyte can block electron conduction more efficiently and has a better promoting effect on protons, which is fundamentally due to the band energy alignment of the interface heterojunction. Our work highlights that LSFNb20 is a high-performance perovskite alternative to monolayer electrolytes.