The invention discloses a method for improving homologous recombination efficiency and recombination virus screening of pseudorabies virus. According to the method, a pseudorabies virus AH strain transfer vector containing an EGFP gene complete expression box is subjected to enzyme digestion linearization, and then subjected to homologous recombination with the pseudorabies virus AH strain; and the obtained recombination virus screening method comprises the steps of separating fluorescent single host cells with pathological changes, and then performing further screening by improved plaque experiment. After recombinant plasmid enzyme is subjected to digestion linearization, the enzyme is subjected to cell transfection; next, virus homologous recombination is performed, so that the homologous recombination efficiency is improved; by virtue of the method in combination with single cell absorption and plaque purification, the screening and purifying time of the recombination viruses is greatly shortened; and therefore, the method is of great significance to improvement of the virus homologous recombination efficiency, has high application prospect in single cell separation, particularly recombination virus screening, and can be widely applied to the recombination virus screening.