An in vitro method for obtaining bamboo mosaic virus (BaMV)-free plantlets of Bambusa oldhamii Munro was developed. BaMV-free meristems were incubated on MS basal medium supplemented with 0.45 μm thidiazuron (TDZ) to induce the development of multiple shoots. Multiple shoot proliferation was higher in stationary liquid culture than on semisolid medium. Cytokinin was the key component for inducing proliferation, and TDZ was the stable and effective cytokinin for proliferation in long-term subcultures. Multiple shoots rooted after 1 month in MS basal medium containing 10.74 to 26.85 μm α-naphthaleneacetic acid with a rooting efficiency of 83%. Healthy, well-developed plantlets were transferred to soil in pots and raised in a greenhouse. Those plants derived from tissue culture were more vigorous than the ones derived from the traditional in vivo vegetative propagation method, air layering. The tissue culture-derived plants could produce the culms after 15 months. Fifteen of 38 plants flowered 2 years after being transplanted to the field.