Dendrobium officinale Kimura et Migo is a famous traditional Chinese medicinal plant. It produces various phytochemicals, particularly polysaccharides, which have nutraceutical and pharmaceutical values. To increase its biomass production and polysaccharide content, our breeding program has generated a series of polyploid cultivars through colchicine treatment of protocorm-like bodies (PLBs). The present study compared two tetraploid cultivars, 201-1-T1 and 201-1-T2, with their diploid parental cultivar, 201-1, in an established in vitro culture system. Tetraploid ‘201-1-T1’ and ‘201-1-T2’ had shorter leaves and shorter and thicker stems and roots, and they produced higher biomass compared with the diploid cultivar. The length and width of stomata significantly increased, but stomatal density decreased in tetraploid cultivars. The PLB induction rates from the stem node explants of the tetraploid cultivars were significantly higher than those of diploid. However, the PLB proliferation of tetraploids was lower than that of the diploid. The mean number of plantlets regenerated from tetraploid PLBs was also lower than that of the diploid after 4 months of culture. Polysaccharide contents in stems, leaves, and roots of 6-month-old tetraploid plantlets were significantly higher than those of diploids. The polysaccharide content in the stem of ‘201-1-T1’ was 12.70%, which was a 2-fold increase compared with the diploid cultivar. Our results showed that chromosome doubling could be a viable way of improving D. officinale in biomass and polysaccharide production.