Hairy roots culture is an effective biological system to solve the shortage of natural resources of Tripterygium wilfordii for the production of the important secondary metabolites. However, there is no enough information about the secondary metabolites in Tripterygium wilfordii hairy root cultures. The present study identified the secondary metabolites in T. wilfordii hairy roots by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS). Subsequently, the nitrogen source and phosphate concentration were optimized to enhance the production of the major active compounds wilforgine and wilforine. Nine compounds including triptolide, wilforgine, wilforine, and so on were identified according to their retention behavior in liquid chromatography and mass spectrometric fragmentation patterns. The Murashige and Skoog (MS) medium was modified for enhancing wilforgine and wilforine production in T. wilfordii hairy root cultures by altering the ratio of NH4+/NO3- and the concentration of phosphate. The results showed that 10:50 mM NH4+/NO3- and 0.3125 mM phosphate were optimal for roots growth and the production of wilforgine and wilforine. After 35 days of cultivation in the modified MS medium containing 10:50 mM NH4+/NO3- and 0.3125 mM phosphate, the maximum production of wilforgine and wilforine was up to 1509.87 mu g.flask(-1) and 578.56 mu g.flask(-1) in T. wilfordii hairy roots separately, a 42 % and 48 % increase over that in the basic MS medium. The present study laid a foundation for the large-scale production of secondary metabolites from T. wilfordii hairy roots.