Abstract: To explore the repairing effect of Dendrobium officinale protein on the injured rat gastric mucosa epithelial cells after the extraction process was optimized. The effects of extraction time, solid-liquid ratio and ammonium sulfate saturation during extraction on the yield of water-soluble total protein of Dendrobium officinale were investigated by single factor, and the optimal extraction process was investigated with the yield of water-soluble total protein of Dendrobium officinale as the response value. Using protein purification technology, DEAE weak anion exchange column chromatography was used to separate the water-soluble total protein of Dendrobium officinale, and rat gastric mucosa epithelial cells (GECs-1) were used to establish an injury model and screen its activity. The structures were characterized by UV scanning, infrared spectroscopy and circular dichroism. The results showed that the optimal extraction process for the water-soluble total protein of Dendrobium officinale was as follows: Extraction time 6 h, solid-liquid ratio 1:27 g/mL, ammonium sulfate saturation 80%, the extraction rate of Dendrobium officinale protein could reach 8.65%±0.13% under these conditions. It was close to the predicted value. Three kinds of proteins were purified from the water-soluble total protein of Dendrobium officinale on the basis of the optimized extraction process. Among them, TPSHP-2 (Dendrobium officinale protein-2) had the maximum absorption peak at 280 nm, which was mainly β-sheet structure, and had the best repair effect on ethanol-injured rat gastric mucosa epithelial cells. The purified TPSHP-2 after the optimized extraction process has a repairing effect on the injured rat gastric mucosa epithelial cells.