To establish cultured cell lines of Artemisia annua L., we used its leaves and stems as explants, MS–based medium and performed three factors of 6–BA, NAA, 2,4–D mass concentration with each factor of 4 levels using L16 (43) orthogonal table for orthogonal test. We also optimized different concentrations of hygromycin and carbenicillin on the growth of the suspension cells and the inhibition effect on Agrobacterium tumefaciens and applied Agrobacterium– mediated method to do genetic transformation of GUS reporter gene and tryptophan monooxygenase iaaM gene in the suspension cells. The results showed that the medium containing MS+6–BA 1.0 mg/L+NAA 0.5 mg/L, 30 mg/L hygromycin and 200 mg/L carbenicillin was optimal and the rate of callus formation is up to 99.33%. And, GUS histochemical staining and PCR analysis of transgenic callus showed that the exogenous gene had been introduced and integrated into the genome of Artemisia annua L., and the genetic transformation system of Artemisia annua L. suspension cells lines was successfully established.