Using the Arnebia euchroma (Royle) Johnst hairy roots induced by Agrobacterium rhizogenes as a testing material,the culture system with two-stage culture method of Arnebia euchroma (Royle) Johnst hairy roots was established for the first time.Factors influencing hairy roots growth such as NH4NO3,different media,speed,temperatures,content of sucrose and factors on hairy roots producing such as method of culture,casein hydrolysate were investigated.The results showed that under the conditions which hairy roots inoculated with MS (no NH4+) liquid medium,the biological production of hairy roots proliferation was doubled that of MS solid medium,and it increased by 8 times cultivated every 15 d.Under the conditions which hairy roots inoculated with 50 mL SH(no NH4+) liquid medium,at (25±1) ℃,(120±5) r/min,the biological production of hairy roots proliferation increased by 9.34 times.The content of sucrose was not critical for hairy roots proliferation.The content of hairy roots shikonin from solid culture and the growth stage could be promoted from about 0.33% to 1.05%.Adding 0.5 g/L casein hydrolysate into the hairy roots,the biological production of hairy roots proliferation could be promoted from about 1.17% to 1.29%,providing a theoretical basis for cultivating large-scale production by applying Arnebia euchroma (Royle) Johnst hairy roots.