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Trends of miniaturized devices and quantum interference electronics lead to the long desire of Fano interference in single-molecule junctions, here, which is successfully demonstrated using the 2,7-di(4-pyridyl)-9,9'-spirobifluorene molecule with a long backbone group and a short side group. Experimentally, the two electrically coupled groups are found to contribute to two blurred degenerate points in the differential conductance mapping. This forms a characteristic non-centrosymmetric double-crossing feature, with distinct temperature response for each crossing. Theoretically, we describe the practical in-junction electron transmission using a new two-tunnelling-channel coupling model and obtain a working formula with a Fano term and a Breit-Wigner term. The formula is shown to provide a good fit for all the mapping data and their temperature dependence in three dimensions, identifying the Fano component. Our work thus forms a complete set of evidence of the Fano interference in a single-molecule junction induced by two-tunnelling-channel coupling transport. Density functional theory calculations are used to corroborate this new physics.