The study objective was to prepare biodegradable branched dextran microspheres encapsulated with His-tagged interferon-alpha (BDM-hIFN-${alpha}$) and evaluate its activity in vitro and in vivo. The glycidyl methacrylate derivatized dextrans (Dex-GMA) as precursor was primarily synthesized by substituting hydroxyl groups of either the branched or linear type of dextran with GMA. Dex-GMA microspheres loaded with hIFN-${alpha}$ was then prepared by the water-in-water emulsion technique. In vitro release and Western blotting experiments demonstrated the retained activity of hIFN-${alpha}$ released from branched dextran microspheres at 24 h by inducing phosphorylation of signal transducer and activator transcription-1 (STAT-1), a down-stream effector of IFN-${alpha}$, in HepG2 cells. Animal data further revealed a peak of plasma levels of IFN-${alpha}$ in rats injected intravenously with BDM-hIFN-${alpha}$ at 10 min post-injection, but a sharp decline at 2 h. High plasma levels of neopterin, a plasma protein induced by IFN-${alpha}$, were also detected in rats injected with BDM-hIFN-${alpha}$ at 10 min post-injection. Notably, plasma levels of neopterin remained high at 4 h, but largely declined thereafter.