Abstract: To explore the best treatment in the main procedures of tissue culture including disinfection, collection period of explants, axillary bud induction, adventitious bud proliferation and rooting culture, and to establish a technical system for tissue culture of Eucalyptus globulus superior seedlings, orthogonal experiment, single-factor experiment and two-factor full factor experiment were used by taking the stem segments of Eucalyptus globulus superior seedlings as explants. The results showed that the optimal treatment for combined disinfection of explants was 75% alcohol disinfection for 20 s, 3% bromo-geramine disinfection for 1 min and 0.05% mercuric chloride disinfection for 6.5 min. The most suitable time to collect explants was in October, during which contamination rate and browning rate were the lowest, survival rate and germination rate were the highest.The best system for axillary bud induction was composed of improving MS medium, 0.5 mg/L KT, 0.4 mg/L 6-BA and 0.5 mg/L NAA, which could significantly increase the germination rate of axillary buds and show stable induction effect. The optimum treatment of adventitious bud proliferation included improving 1/2 MS medium, 0.3 mg/L 6-BA and 0.5 mg/L NAA, which could remarkably increase the proliferation coefficient. The optimal treatment of rooting culture was improving 1/2 MS medium, 0.2 mg/L NAA, 0.5 mg/L IBA and 0.8 mg/L ABT, which could significantly improve rooting rate, increase roots number per plant and the test results were very stable.