The invention discloses a detection method of exogenous avian leukemia virus. The method comprises the following steps: carrying out digestion and dilution on well-grown DF1 cells, carrying out subculture in a cell culture plate, and culturing at 37 DEG C in 5% CO2 for 25-35 minutes; when 45-55% of cells are deposited on the bottom of the cell culture plate, sequentially adding plasma of the detected chicken into every hole of the cell culture plate along the wall, culturing over night, discarding the raw liquor in the cell culture plate, adding maintenance media containing 1% of fetal calf serum, continuing culturing for 7-8 days, and cryopreserving the culture product; and defreezing the culture product, uniformly mixing by slightly blowing and beating, adding the culture product into a P27 antigen reaction plate, detecting the P27 antigen, and judging whether the detected chicken is infected by the exogenous avian leukemia virus. Compared with the traditional process, the method disclosed by the invention can detect the same batch samples at higher positive rate, and has the characteristics of higher detected S/P average value and obvious differences; and the method has equal stability and accuracy, but has higher sensitivity.