The invention discloses a grass carp reovirus S11 gene eukaryotic expression recombinant plasmid preparation method and application thereof in serving as nucleic acid vaccine, and belongs to the technical field of gene engineering and molecular immunology. The preparation method disclosed by the invention is characterized by comprising the steps of extracting viral genome RNA, performing reverse transcription to convert the viral genome RNA into cDNA, amplifying a corresponding DNA sequence out, constructing the DNA sequence to pcDNA-3.1(+) plasmid, converting Escherichia coli DH5alpha, screening out positive clone bacteria containing recombinant plasmid, culturing a lot of the positive bacteria and extracting recombinant plasmid S11-pcDNA3.1 contained in the bacteria. The recombinant plasmid is utilized as nucleic acid vaccine to perform intramuscular injection on the grass carps, and the nucleic acid vaccine enters the muscle cells and expresses VP35 protein of the grass carp reovirus in the muscle cells; thus, fish body immune cell proliferation is stimulated, antiviral related gene expression is up regulated, fish bodies are stimulated to generate antiviral antibodies, and capability of grass carps in resisting grass carp reovirus infection is effectively improved; furthermore, the grass carp reovirus S11 gene eukaryotic expression recombinant plasmid can be used for preventing a grass carp hemorragic disease caused by the grass carp reovirus in aquaculture.