We conducted real-time bioimaging of the hyaluronate-interferon alpha (HA-IFN alpha) conjugate using a biologically inert zwitterionic fluorophore of ZW800-1 for the treatment of hepatitis C virus (HCV) infection. ZW800-1 was labeled on the IFN alpha molecule of the HA-IFN alpha conjugate to investigate its bio distribution and clearance without altering its physicochemical and targeting characteristics. Confocal microscopy clearly visualized the effective in vitro cellular uptake of the HA-IFN alpha conjugate to HepG2 cells. After verifying the biological activity in Daudi cells, we conducted the pharmacokinetic analysis of the HA-IFN alpha conjugate, which confirmed its target-specific delivery to the liver with a prolonged residence time longer than that of PEGylated IFN alpha. In vivo and ex vivo bioimaging of the ZW800-1-labeled HA-IFN alpha conjugate directly showed real-time biodistribution and clearance of the conjugate that are consistent with the biological behaviors analyzed by an enzyme-linked immunosorbent assay. Furthermore, the elevated level of OAS1 mRNA in the liver confirmed in vivo antiviral activity of HA-IFN alpha conjugates. With the data taken together, we could confirm the feasibility of ZW800-1 as a biologically inert fluorophore and target-specific HA-IFN alpha conjugate for the treatment of HCV infection.