The discovery of high-temperature superconductivity in single layer FeSe epitaxially grown on SrTiO3(001) substrates has instigated extensive debate over whether its pairing symmetry is conventional sign-preserving s-wave or unconventional sign-changing. Here, we probe the pairing state for single layer FeSe/SrTiO3 grown by molecular beam epitaxy using scanning tunneling microscopy/spectroscopy. We observe robust in-gap bound states induced by non-magnetic Fe-vacancy defects within the Fe-plane, which exhibit strong spatial electron-hole asymmetry with two-fold symmetry in hole states and four-fold in electron states. The bound states exhibit no energy shift or splitting under an applied magnetic field, consistent with a sign-changing order parameter. This is further confirmed by defect bound state quasiparticle interference that shows a sign-changing behavior with a pair of corresponding peaks at the positive and negative energies near the impurity bound states. Our findings provide unambiguous evidence for a sign-changing pairing symmetry for single layer FeSe/SrTiO3.
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