Abstract: Tripterygium wilfordii Hook. f., adventitious roots were used in vitro to study the effects of inoculation density, liquid medium volume per flask, step-by-step amplification, defoaming agent, macroporous adsorption (MA) resin type, and concentrations of triptolide, wilforgine, and wilforine in roots and medium on adventitious root growth after shake-flask culture. Results showed that an inoculation density of 15 g/L was suitable for the subculture of adventitious roots and accumulation of secondary metabolite. The medium utilization rate was the highest in a 250-mL flask with 100 mL of liquid medium. Adventitious root growth declined slightly with the increase in flask volume. In a 5-L bottle, the adventitious root growth and triptolide, wilforgine, and wilforine concentrations were 91.6%, 91.8%, 91.7%, and 96.9%, respectively, compared to the control， XAD-7 resin markedly enhanced adventitious root growth and secondary metabolite content among the six kinds of MA resin. The adventitious root growth and triptolide, wilforgine, and wilforine content increased 1.2-, 2.9-, 2.4-, and 2.2-fold, respectively, compared to the control when XAD-7 resin was at 0.5 g/bottle. Adventitious root growth and secondary metabolite content all decreased at differing degrees when the defoaming agent was added to the medium. Adventitious root growth and triptolide, wilforgine, and wilforine contents were 85%, 78%, 64%, and 87%, respectively, compared to the control when LX-603 was added to the medium; however, foam was markedly inhibited during the culturing process. This study demonstrated that step-by-step amplification was a feasible approach for adventitious root culture. Furthermore, this research provides the basis for the scale-up culture of T. wilfordii adventitious roots using a bioreactor.