Abstract: Objective: To optimize the purification process of crude polysaccharides from Dendrobium officinale by using response surface methodology. Methods: Through single factor experiments, the effects of (NH₄)₂SO₄ concentration, C₂H₅OH concentration and extraction temperature on the extraction rate and protein removal rate of polysaccharides were investigated, and then a response surface test was performed. The response values of the extraction rate and protein removal rate of polysaccharides were used for statistical analysis. Results: The degree of influence of various factors on the polysaccharides extraction rate and protein removal rate in descending order was extraction temperature > (NH₄)₂SO₄ concentration > C₂H₅OH concentration. Based on the total mass of the two-phase aqueous system, the optimal extraction conditions were as follows: 18.80% (NH₄)₂SO₄, 25.00% C₂H₅OH, extraction temperature was 25 ℃, and the Dendrobium officinale polysaccharide extraction rate and protein removal rate were respectively 94.39% and 91.36%. Conclusion: This method could effectively purify the crude polysaccharides of Dendrobium officinale, and provided a theoretical basis for the development and utilization of Dendrobium officinale polysaccharides.