Aim: We aimed to optimize the steaming process of Pana notoginseng using response surface methodology (RSM). Materials and Methods: The yield and immune activity of steamed P. notoginseng polysaccharides (s-PNPs) was used as the indicators to optimize the steaming parameters: wetting time, steaming time, and temperature. After this, chromatogram and spectroscopy were used to analyze the effect of steaming on the structure of polysaccharides. Then, our study evaluated the immunological activities between s-PNPs and raw P. notoginseng polysaccharides (r-PNPs) in vivo and in vitro. Results: The optimal parameters of processing technology were as follows: wetting time was 25 h, steaming time was 4 h, and steaming temperature was 110°C. The s-PNPs yield reached a maximum of 13.67 ± 0.39%, and the cell proliferation rate was 22.87 ± 0.015% under these conditions. The chemical analysis method showed that the polysaccharide content was slightly increased, the protein content was decreased and the monosaccharide composition analysis indicated that the glucose content in s-PNPs was significantly increased than r-PNPs. Moreover, Fourier transform infrared spectroscopy analysis showed that the functional groups of polysaccharides changed partially between s-PNPs and r-PNPs. In addition, the immunological activity of s-PNPs was better than that of r-PNPs in vivo and in vitro. Conclusion: These studies showed that polysaccharides content was increased, and the immune function of s-PNPs was enhanced after applying steaming, making this approach meaningful for the development and application of P. notoginseng with its immunomodulatory.