The invention discloses a grass carp reovirus spike VP56 protein subunit vaccine, as well as a preparation method and application thereof, and belongs to the technical fields of genetic engineering and molecular immunology. According to the technical scheme, the grass carp reovirus spike VP56 protein subunit vaccine is characterized in that a subunit vaccine protein is coded by a grass carp reovirus S7 gene segment, a base sequence of the grass carp reovirus S7 gene segment is shown as SEQ ID No. 1 in a sequence table, the coded protein is a grass carp reovirus spike VP56 protein subunit vaccine protein, and an amino acid sequence of the subunit vaccine protein is shown as SEQ ID No. 4 in the sequence table. After the subunit vaccine protein is injected into grass carp bodies, immune cellproliferation, antiviral related gene up-regulated expression and specific antibody proliferation of the grass carp bodies can be remarkably stimulated, the capability of grass carps in resisting grass carp reovirus infection is effectively improved, and the grass carp reovirus spike VP56 protein subunit vaccine is used for preventing grass carp hemorrhage caused by viruses in aquaculture.