Interferon alpha (IFN-α) is registered for chronic hepatitis B (CHB) treatment. However, the antiviral mechanism of IFN-α and the biological function of many IFN-α responsive genes have not been fully elucidated. We investigated to determine the regulative effect of IFN-α on toll-like receptor (TLR) 9 signaling in peripheral blood mononuclear cells (PBMCs) from CHB patients in vitro. We examined the changes of expression and function of TLR9 signaling pathway in the PBMCs with different treatment methods and investigated the synergism of IFN-α and TLR9 ligand on antiviral cytokine secretions in vitro. The data showed that, for the TLR9 signaling pathway, IFN-α not only augmented the expressions of TLR9 signal transduction molecules but also activated the TLR9 signal function. This study has clearly demonstrated that the TLR9 ligand could stimulate PBMCs that have been pretreated with IFN-α. Furthermore, the quantity of antiviral cytokines secreted by the pretreated PBMCs was greater than those without pretreatment. The interaction between IFN-α and TLR9 ligand appears to be synergistic. Data revealed IFN-α could influence TLR9 signaling transduction and synergistically improve the immune efficacy of TLR9 ligand against CHB. The present study suggests a potential novel mechanism for the antiviral activity against hepatitis B virus and a new individualized antiviral strategy.