Abstract: To solve the problem of stalk shortening and poor growth of Prunus serrulata‘Royal Burgundy’in vitro, the study of seedling culture in vitro was carried out by using shallow liquid combined with solid culture method. The effect of organic substance, amount and pH value of liquid medium on seedling height, fresh and dried ratio, and the amount of chlorophyll were studied. The results showed that the test tube seedlings grew better in the liquid culture medium, which was 1/2MS+ BA 0.5 mg/L+ IBA 0.5 mg/L+ inositol 300 mg/L, pH value of 6.0, the seedling height was 4.81 cm, the dry matter ratio was 0.088, the chlorophyll content was 2.064 mg/(g·FW). This study improves the tissue culture and propagation technology of P. serrulata‘Royal Burgundy’, and provides technical support for in vitro seedling cultivation.