Polysaccharide is one of the main functional components of Dendrobium officinale. To investigate the in vitro antioxidant and probiotic protection activity of Dendrobium Officinale Polysaccharide (DOP). A polysaccharide from Dendrobium officinale was obtained by water extraction, alcohol precipitation and refined by macroporous resin adsorption in the study. The in vitro antioxidant activity of the DOP was investigated through 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging experiments, ferric ion reducing antioxidant power (FRAP) assays, and HepG2 cell oxidative damage protection experiments. The probiotic protective activity of the DOP was investigated by comparing its effects on the survival rate of Lactobacillus reuteri A9 in gastric juice, intestinal fluid, and bile salt solutions with and without DOP. Results indicated that the DOP is primarily composed of glucose and mannose, with a molar ratio of 0.417:0.567. The Weight-average Molecular weight (Mw) of the DOP is 6.25×105 g·moL-1, and the Number-average Molecular weight (Mn) of the DOP is 1.20×105 g·moL-1. The DOP has a relatively wide molecular weight distribution. The EC50 value of the DOP for scavenging 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) free radicals is 1.53 mg·mL-1, and the FRAP value of the DOP is 0.0172 μmol Fe2+·mg-1, both of which are lower than those of L-Ascorbic Acid (LAA). Results of cell proliferation experiment showed that the DOP can effectively prevent oxidative damage caused by H2O2 to HepG2 cells at a concentration of 200, 400 and 800 μg·mL-1, and are better than those of LAA. The DOP effectively improved the survival rates of Lactobacillus reuteri A9, in simulated gastric fluid, simulated intestinal fluid, and bile salt solution. Demonstrating that the DOP possesses favorable lactic acid bacteria protective activity. This study provides reference for the development and application of DOP, and the development of new probiotic protectants.