Objective To determine the content of polysaccharides of Rhodobryum giganteum from different regions in Yunnan province, China. Methods The optimal process of extraction of freeze-dried powder from R. giganteum was determined by orthogonal experiments. The contents of polysaccharides were determined by phenol-sulfuric acid method and investigation of methodological. Results The optimal process of extraction with water was determined as follows. The number of times has been decocted as 2 times, the first time with 24 times the amount of water for 30 minutes, and the second time with 22 times the amount of water for 25 minutes. In the range of concentration of 0.01~0.05 mg/mL, glucose had a good linear relationship with its absorption, and the linear regression equation was y = 14.29x+0.2097(R2 = 0.9994). The average recovery of glucose was 98.8%. Moreover, the contents of polysaccharides were between 15.75% and 65.96% in water decoction of R. giganteum for 15 batches. Conclusion The operation is simple for the extraction method, and its results are characterized by high accuracy and reliability, which can be used to determine the content of polysaccharides for R. giganteum.