Background: Chromolaena odorata Linn. (CO) is a perennial herb that is enriched with flavonoids and exhibits immune regulatory activities. For these characteristics, CO can be used as a potential immunoregulator based on the immunosuppression state. Objectives: The aim of this study was to assess the effects of flavonoids extracted from CO (FCO) on immune regulation and evaluate their mechanism of action by network pharmacology (NP), followed by in vivo confirmation. Materials and Methods: FCO were ultrasonically extracted through immersion in alcohol. The potential targets were predicted using a “FCO–immunosuppression–target” network. When the functional enrichment analyses were conducted, a mice model was employed to demonstrate the effects. The hematological indexes and serum levels of immunoglobulin G (IgG), immunoglobulin M (IgM), interferon-γ (INF-γ), interleukin 2 (IL-2), and tumor necrosis factor-α (TNF-α) were measured. The variations observed in immune organs and the changes in expressions of INF-γ, T-box transcription factor 21 (Tbx-21), and GATA Binding Protein 3 (GATA3) were reported. Results: NP results showed that a total of 198 targets of FCO were involved in immunosuppression. As indicated by the functional analysis results, FCO impacted T helper (Th) cell differentiation, which might be a vital functional pathway underlying its immune regulatory effects. During animal experiments, the values of hematological indexes, serum levels of IgG, IgM, and TNF-α, and the immune organ indexes increased in FCO groups, and the relative mRNA expressions of INF-γ and Tbx-21 and less damage of the spleen and thymus were reported. Conclusion: FCO impacts Th1 and Th2 differentiation pathways and assists in immunosuppression by regulating the secretion of various cytokines and the expression of associated genes, which demonstrates FCO as a promising natural immunoregulator.