The invention relates to the field of biotechnology, in particular to a method for rapidly screening the recombinant fowlpox virus by means of a CRISPR/Cas9 system and application thereof. The method comprises the steps of designing an sgRNA (short guide RNA) double-stranded oligonucleotide sequence of a target gene, ligating the sequence with a linearized plasmid vector to obtain an sgRNA expression vector, co-transfecting chicken embryo fibroblasts (CEF) with the sgRNA expression vector, recombinant fowlpox virus plasmids expressing a foreign gene and the fowlpox virus, and conducting purification and verifying the purification effect. By means of the CRISPR/Cas9 method, the number of recombinant fowlpox virus screening generations is reduced to 3-4, the efficiency of vaccine production is greatly improved, operation is easy, and the production cost is reduced.