Abstract: Newly born stems with axillary buds of eminent Acacia melanoxylon (AMY12004) were used as explants and cultured on MS after sterilization. The aseptic leaf, petiole and stem of germinated axillaries were used to induce callus, and high-frequency regeneration systems were established via indirect organogenesis. The optimal material for callus induction was the stem, the optimal medium for induction was MS+1.5 mg·L^–16-BA+0.2 mg·L^–1NAA+3 sucrose, and the optimal medium for redifferentiation was MS+2.0 mg·L^–16-BA+0.5 mg·L^–1NAA+3%sucrose. The callus induction rate, redifferentiation rate and regeneration coefficient was 93.33%, 79.17% and 9.58, respectively. The optimal rooting medium was MS+0.5 mg·L^–1IBA+0.5 mg·L^–1NAA+4%sucrose and the highest rooting rate was 96.05%; the transplant survival rate was 81.40%.