Recently, a super uranyl binding protein (SUP) was developed, which exhibits excellent sensitivity/selectivity to bind uranyl ions. It can be immobilized onto a surface in sensing devices to detect uranyl ions. Here, sum frequency generation (SFG) vibrational spectroscopy was applied to probe the interfacial structures of surface-immobilized SUP. The collected SFG spectra were compared to the calculated orientation-dependent SUP SFG spectra using a one-excitonic Hamiltonian approach based on the SUP crystal structures to deduce the most likely surface-immobilized SUP orientation(s). Furthermore, discrete molecular dynamics (DMD) simulation was applied to refine the surface-immobilized SUP conformations and orientations. The immobilized SUP structures calculated from DMD simulations confirmed the SUP orientations obtained from SFG data analyzed based on the crystal structures and were then used for a new round of SFG orientation analysis to more accurately determine the interfacial orientations and conformations of immobilized SUP before and after uranyl ion binding, providing an in-depth understanding of molecular interactions between SUP and the surface and the effect of uranyl ion binding on the SUP interfacial structures. We believe that the developed method of combining SFG measurements, DMD simulation, and Hamiltonian data analysis approach is widely applicable to study biomolecules at solid/liquid interfaces.