Ebola virus (EBOV) is responsible for several outbreaks of hemorrhagic fever with high mortality, raising great public concern. Several cell surface receptors have been identified to mediate EBOV binding and internalization, including phosphatidylserine (PS) receptors (TIM-1) and C-type lectin receptors (DC-SIGNR). However, the role of TIM-1 during early cell surface binding remains elusive and in particular whether TIM-1 acts as a specific receptor for EBOV. Here, we used force–distance curve-based atomic force microscopy (FD-based AFM) to quantify the binding between TIM-1/DC-SIGNR and EBOV glycoprotein (GP) and observed that both receptors specifically bind to GP with high-affinity. Since TIM-1 can also directly interact with PS at the single-molecule level, we also confirmed that TIM-1 acts as dual-function receptors of EBOV. These results highlight the direct involvement of multiple high-affinity receptors in the first steps of binding to cell surfaces, thus offering new perspectives for the development of anti-EBOV therapeutic molecules.