Three polysaccharide fractions from bamboo shoot (Chimonobambusa quadrangularis), CPS70, CPS75, and CPS80, were prepared using a final ethanol concentration of 70%, 75%, and 80% in the precipitation process. In vitro digestibility and the prebiotic activity of CPS70, CPS75, and CPS80 were evaluated and compared. The results indicated that all three of the CPS fractions exhibit a high degree of nondigestibility to human gastric juice (>98.5%) or α‐amylase hydrolysis (>94.5%). Compared with the blank control, the three CPS fractions could not only significantly (p < .05) stimulate the proliferation of B. adolescentis, B. infantis, B. bifidum, and L. acidophilus, but also significantly (p < .05) enhance the production of lactic, acetic, propionic, and butyric acids when these polysaccharides were added as alternative carbon sources to glucose during the in vitro fermentation of four probiotics. Furthermore, when comparing the three CPS fractions, CPS75 displayed the strongest prebiotic potential, as this polysaccharide had the strongest effect on the proliferation of probiotic bacteria as well as the greatest effect on SCFAs production. These results demonstrated that the concentration of ethanol used during the precipitation process has a significant impact on the prebiotic activity of CPS. Practical applications Ethanol precipitation is the first step when extracting polysaccharides from aqueous extracts as it is simple, rapid, and easy to carry out. This study focuses on how different concentrations of ethanol used in the precipitation process affect the prebiotic potential of bamboo shoot (Chimonobambusa quadrangularis) polysaccharides (CPS). The result indicated that the concentration of ethanol used during the precipitation process has a significant impact on the prebiotic activity of CPS. To our knowledge, it is the first to evaluate the effects of the concentration of ethanol during the process of precipitation on prebiotic potential of polysaccharides, which can subsequently be applied to the optimization of ethanol concentration when precipitating natural polysaccharides for the purpose of in vitro fermentation.