In order to research the role of soybean oligosaccharides (SBOSs) on improvements in the microenvironment of intestinal flora and immune function of cyclophosphamide (CTX) immunosuppressive mice. Via giving intragastric administration of Soybean oligosaccharide (SBOS) at the low dose (50/(kg·BW)/d), the middle dose (200 mg/(kg·BW)/d) and the high dose (500 mg/(kg·BW)/d) partly once a day, which is also 28 days in a row. At the same time, (SBOS) mice in the drug group and (CG) mice in the positive control group were given intraabdominal injection of CTX (200 mg/kg/d).The immunosuppressive mouse model (CY) was established after 72 h in the model group and the positive control group (CG) was given intragastric administration of levamisole hydrochloric acid (LMS) for 3 days, with the data of 80ug/kg/d after injection of CTX (for actually 72 h). On the 8th, 15th and 22nd day, the number of Bifidobacterium, Lactobacillus, Enterococcus and Clostridium perfringens m in the feces of mice in each dose of drug group were determined. After the test resulted, the cellular immune function, humoral immune function, monocyte/macrophage function, NK cell activity and cytokine secretion (tumor necrosis factor-α, interferon-gamma and IL-4) were measured in immunosuppressive mice each group. The results showed that 200 mg/(kg BW) soybean oligosaccharide could significantly promote the proliferation and inhibit the increase of Enterococcus in immunosuppressive mice. The soybean oligosaccharide of 500 mg/(kg BW) could dramatically promote the proliferation of both Bifidobacillus and Lactobacillus, and also inhibit the increase of both Enterobacteriaceae and Enterococcus in immunosuppressive mice. The regulatory function of SBOS on intestinal flora was positive. Soybean oligosaccharide (500 mg/(kg BW) could significantly promote the proliferation of Bifidobacillus and Lactobacillus in immunosuppressive mice and inhibit the increase of Enterococcus and Enterococcus. The proliferation of spleen lymphocytes induced by ConA, LPS in immunosuppressive mice was dose-dependent. But it was still lower than that of the normal group (CG0) (p > 0.05). The serum hemolysin level of immunosuppressive mice was significantly increased in each dose group (p < 0.05), and the level of antibody forming cells in spleen cells of each dose group was significantly increased (P < 0.05), and the level of antibody forming cells in spleen cells of each dose group was significantly higher than that of low dose group (p < 0.005), and the level of serum hemolysin in immunosuppressive mice was significantly increased in each dose group (p < 0.05). In the detection of immune effector cell activity in immunosuppressive mice, the phagocytic function of macrophages in high dose group and the natural killing activity of spleen NK cells in high dose drug group were significantly increased, which were not significantly different from those in positive control group (P < 0.05), but the expression of TNF-α, INF-γ and IL-4 cytokines in serum was increased in a dose dependent manner (p < 0.05). In conclusion, soybean oligosaccharide can significantly increase the diversity of intestinal microecology, increase the number of intestinal beneficial bacteria, has a correlation with the proliferation of Bifidobacterium and Lactobacillus in the intestinal tract, and inhibit the proliferation of harmful bacteria. The results showed that SBOS had a direct effect on the proliferation of intestinal flora under immunosuppression. Based on the improvement of intestinal microenvironment in immunosuppressive mice by soybean oligosaccharide for 25 days, the results showed that compared with the positive control group, the nonspecific and specific immunity of immunosuppressive mice in the drug group had a regulatory effect, which improved the phagocytic function of monocytes/macrophages, developed the level of antibody forming cells, enhanced the standard of the killing activity of NK cells, and promoted the expression of cytokines as well. Compared with the model group, the transformation and proliferation of spleen lymphocytes in the high and middle dose groups were remarkably increased, but all of the indexes did not reach the level of the normal blank group. By studying the improvement of intestinal microenvironment in immunosuppressive mice, to some extent, it is concluded that the proliferation of intestinal flora can improve the immunomodulatory function of the body, but it still lowers the normal immune degree, which reflects the immunomodulatory effect of the body on the stimulation of continuous external intake. The results demonstrate that the immunomodulatory ability of immunosuppressive body was insensitive to SBOS and provided a theoretical basis for the study of health care function of intestinal microenvironment improvement when SBOS acted on abnormal immune function. The results also improved the practical application value of SBOS.