The invention discloses a purification method of recombinant viruses with fluorescent markers. The purification method comprises the following steps: 1, goat pox viruses with KLP2 genes being lost arerecombined, specifically, (1) sheep testicular primary cells are cultured, a cell plate is spread, after cell culture reaches 80% or so, the cells are infected through goat pox virus vaccine strains,and (2) virus recombination is conducted; and 2, the recombinant viruses are purified, specifically, (1) the recombinant viruses with fluorescence are picked, (2) the viruses are purified, (3) purification is continued, specifically, the viruses, with the fluorescence, picked in the previous step are repeatedly subjected to freeze thawing, then according to operation of (2) in the step 2, dilution and inoculation are conducted, then picking is conducted, and generally after 3-4 generations, the purified recombinant viruses can be obtained.