In this paper, differences of phenotypic traits and chemical constituents were compared in 11 groups of Dendrobium nobile samples. Several phenotypic parameters, such as the plant height, leaf length and width, stem length, stem diameter and node number, and water content were measured in five tissue culture materials with the same genotype and 2-year-old fresh stems of six landraces. Contents of dendrobium alkaloids, polysaccharide, total protein, crude fiber and water were determined by acidic dye colorimetry, anthrone-sulfuricmethod, UV spectrophotometry, filter bag technology, and drying method, respectively. After comparing the phenotypic traits, there were differences in terms of phenotypic traits for tissue materials cultivated by different culture medium. Specifically, Wanglong No. 4 contained the highest dendrobium alkaloids (0.2627%) and polysaccharide (6.86%). The callus culture by medium A contained the highest total protein content. However, the water content in fibrous Wanglong No. 1 was the least with 83.07%. We obtained that the phenotype was significantly distinctive due to different cultivation conditions, especially the dendrobium alkaloids, polysaccharide, total protein and crude fiber. Improving the cultivation condition was beneficial to enhance the chemical constituents content. This study provided an experimental basis to improve the cultivation protocol and offered a reference to change the medicinal component contents in D. nobile.