【Objective】 Mytilaria laosensis is an evergreen, broad-leaved tree with a wide range of suitable habitats and good economic and ecological protection. The seedling is mostly used for cultivation. Grafting and cutting techniques are still experimental, and few reports on tissue culture techniques exist. Therefore, it is of great significance to establish an efficient tissue culture technology system for M. laosensis and to provide technical support for promoting its clone in the future. 【Method】 With the semi-lignified bud at the base of the superior tree as explants, through the processes of primary axillary bud induction, secondary proliferation culture, rooting culture, the optimal explant collection time, the basic breeding and rooting culture medium, and the type and concentration ratio of plant growth regulator were studied. 【Result】 There were significant differences in the effect of different sampling times on the induction of M. laosensis. The best induction of explants was collected in October 2022, with a survival rate of 32.38%. Treatment with different hormone concentrations significantly affected the time required for bud germination and the growth status of explants. The optimal formula for axillary bud induction was MS+6-BA 0.5 mg/L+NAA 0.02 mg/L, the induction rate was 100%, and the germination time was 4.07 d. The selection of different basic mediums significantly affected the proliferation of M. laosensis. MX medium had the highest average shoot length (4.47 cm) and the largest proliferation coefficient (4.41), respectively, which were significantly higher than those of other basic media (1/2 MS, MS, DKW, WPM), and the proliferation buds grew strongly, increased quickly and had a good effect. Different hormone types and treatment concentrations significantly affected the proliferation and rooting effects of M. laosensis. The optimal breeding medium was MX (modified WPM) + 6-BA 1.0 mg/L+NAA 0.05 mg/L, and the proliferation coefficient reached 4.94. The buds grow vigorously. The optimal rooting medium was MX+NAA 0.3 mg/L, the rooting rate was 100%, the average root number was 13.18, root growth was robust, and the transplanting survival rate reached 92.3%. 【Conclusion】 The induction survival rate of M. laosensis explants was related to the location and time of material collection. October was the best time to collect explant material in Guangdong. In the case of a low proliferation coefficient of the MS basic medium, switching to an MX (modified WPM) medium could significantly increase the proliferation succession factor. Then, the induction medium, breeding medium, and rooting medium were optimized, which helped establish the tissue culture system of the superior tree of M. laosensis and improved the quality and efficiency of the tissue culture.