The invention relates to a culture method of walnut plantlets, in particular to a method for treating walnut tissue culture stem segments inside test tubes and rooting outside the test tubes. The invention aims at solving such problems that restrict the industrial plantlet culture of the walnuts as low survival rate and rooting difficulty in propagation by tissue culture since variation is apt togenerate in propagation by seedling of the walnuts and connection parts are polluted easily in propagation by grafting. The concrete steps are as follows: (1) obtaining of the walnut tissue culture stem segments; (2) treatment of the tissue culture stem segments inside the test tubes; (3) cuttage of the tissue culture stem segments outside the test tubes; and (4) management and transplanting of the tissue culture stem segments after cuttage. The method combines the walnut tissue culture, the treatment of the tissue culture stem segments inside the test tubes and the cuttage and the rooting outside the test tubes, so that a high-efficient cuttage rooting and seedling culture technology for the walnut tissue culture stem segments is created, the cuttage rooting rate is up to above 80% and the transplanting survival rate is up to above 75%. The technology has the characteristics of saving cost, shortening seedling culture period, improving the transplanting survival rate, realizing annual production and enabling the seedlings to be regular and the technology has great promotional value.