A hairy roots culture of Stephania suberosa was established using Agrobacterium rhizogenes ATCC15834. The production of dicentrine was found to be (8.92 +/- 0.07) mg/g dry wt on day 35 of culture. Effects of sucrose content, tyrosine, and medium strength on growth and dicentrine production of S. suberosa were investigated. 6% (w/v) sucrose was an optimum content for the growth and dicentrine accumulation in S. suberosa hairy roots. The utilization of a precursor from tyrosine feeding enhanced the dicentrine production. The medium with 1.0 mM of tyrosine had the highest effect on dicentrine accumulation in hairy roots at day 40 of culture [(14.73 +/- 0.47) mg/g dry wt]. In addition, 1/4 Murashige and Skoog medium was suitable for biomass and dicentrine production in hairy roots. This culture system has a potential to produce dicentrine from hairy roots of S. suberosa.