Abstract: Objective: Active fractions with immunomodulatory effects from the stems and leaves of Astragalus membranaceus (AMSL) were investigated and its chemical constituents were analyzed. Methods: The immunosuppression mice models were established by injection of cyclophosphamide, and the effects of several AMSL extracts on the immune response of mice were investigated, including crude extract, petroleum ether fraction, chloroform fraction, ethyl acetate fraction, n-butanol fraction and water fraction. Meanwhile, the control group, model group, and positive group were included. Hematoxylin-eosin staining was used to examine the spleen tissue morphology of mice after 28 d of continuous administration, and the immune organ indexes, immune cell capabilities, and levels of immune components in each group were analyzed. Furthermore, the components in the active fraction of AMSL were identified through ultra-performance liquid chromatography/ion mobility quadrupole time-of-flight mass spectrometry (UPLC/IM-QTOF-MS). Results: The n-butanol fraction from AMSL was proved to show significant immunomodulatory activity. In comparison to the model group, the growth rate of body weight, immune organ indexes, splenic lymphocyte proliferation index, macrophage phagocytic capacity, delayed allergic reaction severity, and serum hemolysin level in mice were significantly enhanced (P<0.05) in the group treated by this active fraction. Besides, the serum levels of immunoglobulin A, interleukin-6, interferon-γ and tumor necrosis factor-α were also significantly increased by 39.96%, 15.87%, 16.22% and 47.68% respectively after the intervention of this fraction (P<0.05), which was revealed to be primarily composed of such substances as flavonoids, saponins, and phenolic acids elucidated by UPLC/IM-QTOF-MS. Conclusion: The active fraction with immunomodulatory effects from AMSL was the n-butanol fraction, which established a basis for further study on the immunoregulatory activity of AMSL.