Ethanol fractional precipitation can initially separate polysaccharides according to the structure, which exhibits strong correlation with the biological activities. This study aimed to investigate the impact of varying ethanol concentrations on the structural characteristics, and the antitumor and antioxidant activities of polysaccharides derived from Dendrobium officinale through ethanol fractional precipitation, as well as their internal relationships. The polysaccharides acquired by absolute alcohol additions at a final liquor-ethanol volume ratio of 1:1, 1:2, and 1:4 were named DOP-1, DOP-2, and DOP-4, and the supernatant was named DOP-S. The results of the structural analysis revealed that the increase in ethanol concentrations resulted in a reduction in the molecular weights and the acetylation degree of the polysaccharides, as well as a decrease in mannose content and an increase in glucose content. In vitro experiments demonstrated that DOP-S exhibited optimal antitumor and antioxidant activities. Animal experiments further confirmed that DOP-S suppressed the growth of solid tumors significantly, enhanced lymphocytes, mediated immune ability, and improved the activity of antioxidant enzymes. These findings would establish a theoretical foundation and provide technical support for further advances and applications of polysaccharides derived from D. officinale in the fields of food and medicine.