Combinatory enhancement of innate and adaptive immune responses is a promising strategy in immunotherapeutic drug development. Bifunctional macromolecules that simultaneously target two mechanisms may provide additional advantages over the combination of targeting two single pathways. Interferon alpha (IFNα) has been used clinically against viral infection such as the chronic infection of hepatitis B virus (CHB) as well as some types of cancers. OX40 is a costimulatory immune checkpoint molecule involved in the activation of T lymphocytes. To test whether simultaneously activating IFNα and OX40 signaling pathway could produce a synergistic therapeutic effect on CHB and tumors, we designed a bifunctional fusion protein composed of a mouse OX40 agonistic monoclonal antibody (OX86) and a mouse IFNα4, joined by a flexible (GGGGS) linker. This fusion protein, termed OX86-IFN, can activate both IFNα and OX40. We demonstrated that OX86-IFN could effectively activate T lymphocytes in the peripheral blood of mice. Furthermore, we showed that OX86-IFN had superior therapeutic effect to monotherapies in HBV hydrodynamic transfection and syngeneic tumor models. Collectively, our data suggests that simultaneously targeting interferon and OX40 signaling pathways by bifunctional molecule OX86-IFN elicits potent antiviral and antitumor activities, which could provide a new strategy in developing therapeutic agents against viral infection and tumors.