Dendrobium nobile Lindl. (D.nobile) is a perennial herb of the orchid family and is commonly used in traditional Chinese medicine. Due to the scarcity of D.nobile resources, the market faces a shortage. To improve cultivation efficiency and reduce costs, a temporary immersion bioreactor system (TIBS) is used to meet market demand. The optimal cultivation conditions for D.nobile tissue culture seedlings have been determined through experimental research. These conditions include a liquid medium consisting of 1/2 MS, 30 g/L sucrose, 80 g/L potato juice, 0.5 mg/L NAA, 60 g/L banana juice, and a pH of 6.0. The optimal inoculation volume of TIBS per canister was 22% plant/V D.nobile seedlings, and the optimal submergence frequency of TIBS was set at 5 min/8 h, and the optimal submergence time was set at 6 min/6 h. It was also observed that the endophytic fungus MD33 had a significant impact on plant growth and the dendrobine-type total alkaloids (DTTAs) content, and the accumulation of DTTAs content of D.nobile tissue culture seedlings was significantly higher than that of the control group at 25 days, with an increase of 40.61% (P < 0.001). This study demonstrates that utilising TIBS for cultivation and co-cultivating with endophytic fungus significantly increased the biomass and bioactivity of D.nobile tissue culture seedlings.