Oxymatrine, extracted from the herb Sophora alopecuraides L., was investigated to determine its anti-HBV immunomodulatory mechanism in vitro. Human peripheral lymphocytes were isolated from heparinized whole blood from 48 chronic hepatitis B (CHB) patients. The lymphocytes from each patient were divided into two groups according to pretreatment or no pretreatment with Oxymatrine in vitro. We examined the changes of expression and function of the toll-like receptor 9 (TLR9) signal transduction pathway in the peripheral lymphocytes with different treatment methods and investigated the synergism of Oxymatrine and the TLR9 ligand on antiviral cytokine secretions in vitro. The data showed Oxymatrine could induce antiviral cytokine secretions directly from the peripheral lymphocytes. For the TLR9 signal pathway, Oxymatrine not only augmented the expressions of TLR9 signal transduction molecules, but also activated the TLR9 signal function. This study has clearly demonstrated that TLR9 ligand could stimulate peripheral lymphocytes that have been pretreated with Oxymatrine. Furthermore, the quantity of antiviral cytokines secreted by the pretreated lymphocytes was greater than that of those without pretreatment. The interaction between the Oxymatrine and the TLR9 ligand appears to be synergistic. This study suggests Oxymatrine could be a strong immunomodulator, influence TLR9 signaling transduction, and synergistically improve the immune efficacy of the TLR9 ligand against CHB.