A method of analyzing protein-protein interactions at a single molecular level is disclosed. The method of analyzing the interactions between first proteins and second proteins at the single molecular level includes: preparing at least two substrates, in which first protein-binding molecules that are biomolecules to be bound to the first proteins are attached to each of the substrates; inducing binding between the first proteins and the first protein-binding molecules on the first substrate and the second substrate, respectively, by supplying the first proteins included in the control group-cell to the first substrate among the two substrates and supplying the first proteins included in the experimental group-cell to the second substrate among the two substrates; supplying cell lysates of cells including the marker-tagged second proteins to the first substrate and the second substrate, respectively, when the first proteins and the first protein-binding molecules are bound to the first substrate and the second substrate, respectively; and comparing and analyzing the interactions between the first proteins and the second proteins on the first substrate and the second substrate in the supply of the cell lysates to the first substrate and the second substrate, respectively. For observing the interactions between the first proteins and the second proteins, the state of each cell and activation levels of the first proteins can be compared and analyzed by comparing after varying of a type of cells supplying the first proteins.