Abstract: The properties of Magnolia officinlis resources are attractive and in high utilization. It is in great demand at the market. Magnolia officinlis in natural are scarce resources, to force the people to create Magnolia officinlis plantation, but the low-yield of the Magnolia officinlis seed will be a bottleneck to seedling. The callus initiation culture of Magnolia officinlis shoots in the test will provide a reference for rapid propagation system of tissue culture. According to analysis and LSD multiple comparison analysis, the best medium, optimal hormone and culture sterilization conditions to start culturing Magnolia officinlis seedlings callus were studied by completely randomized experimental design. It indicated: the basic medium B5 was better, the lower NAA concentration could promote to initiation culture, and the higher was benefical to form callus; when added different (6-BA) and (2,4-D) into medium B5, the differentiation rate of callus was highest in the medium (B5 + 6-BA 3.0 mg/L + 2,4-D 3.0 mg/L), the buds the medium (B5 + 6-BA 4.5 mg/L + 2,4-D 2.0 mg/L); when added 6-BA and NAA into medium B5, the medium (B5 + 6-BA 4.5 mg/L + NAA 1.0 mg/L) was best to differentiate callus, the medium (B5 + 6-BA 5.0 mg/L + NAA 1.0 mg/L) the buds. Considering hormone interaction, the mediume (B5 +5.0 mg/L + NAA 1.0 mg/L) was the best to start culturing Magnolia officinlis seedlings callus. The study will provide a reference for rapid propagation system of Magnolia officinlis seedlings tissue culture.