The disclosure provides a recombinant nucleic acid of Seneca valley virus, a recombinant vaccine strain and preparation method and use thereof, and relates to the technical field of genetic engineering. The disclosure provides the recombinant nucleic acid of Seneca valley virus, recombinant Seneca valley virus comprising the recombinant nucleic acid, recombinant Seneca valley virus encoded by the recombinant nucleic acid, recombinant Seneca valley virus vaccine strain comprising the recombinant Seneca valley virus and preparation method and use thereof. According to the disclosure, a vaccine strain characterized by high antigen production capacity, remarkably reduced pathogenicity (even having no pathogenicity to pigs), strong antibody induction activity, high immune protection rate is prepared. The vaccine strain remarkably improves the biological safety and can be used for preventing and controlling Seneca valley virus in China and the neighboring countries.