The aim of the first part in this study was to investigate the suitable culture conditions for shoot multiplication and growth of single node culture of Stevia rebaudiana in vitro by supplementing auxin, cytokinin and chitosan in the culture media. The uses of IAA (1 and 2 mg/L) in combination with either BA or kinetin (0.25, 0.5, 0.75 and 1 mg/L) were compared with BA (0.5, 1, 1.5 and 2 mg/L) or kinetin (6, 12 and 18 mg/L) alone in MS medium. As the result, no significant difference was found in shoot multiplication among the treatments using plant growth regulators and the control. However, the use of BA at the concentration of 2 mg/L could increase the fresh and dry weight of shoot to 2.8 and 2.7 times of the control, respectively. Application of chitosan P80 and P90 at the concentration of 10 and 5 mg/L was found to increase average number of shoots per node to 2.00 ± 0.00, which tended to be higher than the other treatments. In addition, it was found that O80 chitosan at the concentration of 5 mg/L could stimulate shoot growth by increasing the shoot length from 7.0 ± 0.89 mm of the control to 11.13 ± 1.76 mm which caused the increase of average fresh and dry weight of shoot to 2.3 and 2.2 fold of the control, respectively. Apart from that, the same treatment also tended to induce more number of roots than the control did. In the second part of the study, the stevioside content in cultured tissue was analyzed. Low stevioside content was found in the treatments supplemented with plant growth regulators while stevioside content in the treatments using 40 mg/L P90, 20 mg/L O80 and 10 mg/L O90 chitosan were significantly increased to 1.3 times of the control.