The invention discloses a Vsig4 nano-antibody and an epitope identification method and application thereof and belongs to the technical field of biological pharmacy. The invention further discloses a gene sequence for encoding the nano-antibody, an expression vector and a host cell capable of expressing the nano-antibody and an epitope obtained by combining the nano-antibody, a mouse source Vsig4 and a human source Vsig4. According to the gene sequence, the expression vector and the host cell of the nano-antibody, the nano-antibody can be efficiently expressed in escherichia coli, stable and uniform Vsig4 nano-antibody protein with high affinity is optimized and obtained, and the nano-antibody is applied to research and development of a Vsig4 molecular detection reagent and serves as an antagonist for blocking combination of Vsig4 protein molecules and a complement C3b.