The invention discloses a subculture method of Cornus walteri Wanger. tissue culture seedlings. The subculture method includes: inoculating the bud tussocks, smaller than 1cm, of the sterile Cornus walteri Wanger. tissue culture seedlings satisfying subculture requirements into a proliferation culture medium, inoculating robust buds, not smaller than 1cm, of the tissue culture seedlings into an elongation culture medium, wherein the proliferation requirements of proliferation expanding propagation and high branches and robust seedlings are satisfied at the same. The subculture method has the advantages that the subculture method which is high in operability and combines proliferation and elongation growth is built by using different culture media, the average proliferation coefficient is above 10, average plant height is above 3.5cm, the tissue culture seedlings grow well, proliferation coefficient is stable after successive culture, and the subculture method is applicable to the large-scale production of the Cornus walteri Wanger. tissue culture seedlings and good in economic and social benefits.