In order to figure out the best experimental conditions and to establish a complete and efficient system for rapid propagation of blueberry, a series of experiments were conducted on explant sterilization, buds induction, subculture of tissue–cultured seedlings, rooting and transplanting of tissue–cultured seedlings by employed twigs of ‘South Golden’, a variety of southern highbush blueberries, stems with axillary buds were used as explants. The results indicated that the explants soaked in 5% NaClO solution for 10 min, then in 0.1% HgCl2 solution for 7 min displayed the best effect in disinfection. Modified WPM with 4.0 mg/L ZT was the best medium for inducing buds, and the multiplication coefficient reached to 3.86, the average height of buds was 19.4 mm; modified WPM with 1.0 mg/L ZT was the best subculture medium, the multiplication coefficient was 1.82 and the average height of seedlings on this medium reached to 44.7 mm; modified 1/4WPM with 0.5 mg/L IBA and 1.0 mg/L NAA was considered as the best medium for inducing roots of tissue–cultured seedlings; vermiculite mixed with peat soil (the proportion of them was 1∶1) was the most appropriate substrate for transplanting and cultivating tissue–cultured seedling, the survival rate reached up to 96.95%.