Triptolide, an important representative active compound of Tripterygium wilfordii, is hopeful to be developed into modern drug. However, the content of triptolide in natural plants is very low, which seriously restricts its development in the pharmaceutical industry. As a perennial woody vine, research on the genetic transformation of T. wilfordii is challenging and slow, which limits the process of improving triptolide content through biotechnology. In this study, the concentrations and combinations of growth regulators for T. wilfordii callus induction and regeneration were firstly optimized. The 2,4-dichlorophenoxyacetic acid (1 mg/L) was efficient to induce callus formation from leaf explants, while 2.0 mg/L 6-benzylaminopurine with 0.1 mg/L α-naphthaleneacetic acid had the best effect on regeneration from callus. Subsequently, the effect of different concentrations of screening agent (kanamycin or hygromycin B) on callus induction rate was determined. Then, Agrobacterium tumefaciens mediated genetic transformation was established and validated by two different plant expression vectors, pCambia1301 and pJCV53, corresponding to β-glucuronidase histochemical assay and monomeric red fluorescent protein fluorescence detection, respectively. It took 3–4 months from A. tumefaciens infection to complete transgenic plant regeneration. Based on this transformation system of T. wilfordii, the overexpression of miltiradiene synthase gene (TwMS) was performed. Real-time quantitative PCR analysis indicated that the expression levels of TwMS were highly up regulated in the transgenic plants compared with control line. The detection of high-performance liquid chromatography showed that overexpression of TwMS resulted in a 1.20–1.51-fold increase of triptolide in the transgenic lines. These results indicated that this efficient A. tumefaciens mediated genetic transformation provides a useful tool for future T. wilfordii genetic engineering studies to enhance the accumulation of triptolide in this economically important medicinal plant.