Provided are a PR8 recombinant influenza virus and the preparation method and use thereof. The recombinant influenza virus contains an HA and/or NA gene of Hl, H3, H4, H5, H6, H7, H9 or H10 subtype influenza virus, and 6 internal genes of PR8 virus — PB1, PB2, PA, M, NS and NP genes, wherein the NS and/or NP gene have the following point mutations: the NS2 protein encoded by the NS gene has an E67S point mutation, E47S point mutation, or E67S/E47S point mutation, and the NP protein encoded by the NP gene has a G132A point mutation. The HA protein and/or NA gene can be highly expressed by constructing a recombinant plasmid and then co-transfecting the plasmid into cell nuclei of a SPF chicken embryo to amplify the abovementioned PR8 recombinant influenza virus, which can be used in large-scale preparation of influenza vaccine.
