The purpose of this study was to prepare and characterize virosome containing envelope proteins of the avian influenza (H5N1) virus. The virosome was prepared by the solubilization of virus with octaethyleneglycol mono (n-dodecyl) ether (C12E8) followed by detergent removal with SM2 Bio-Beads. Biochemical analysis by SDS-PAGE and western blotting, indicated that avian influenza H5N1 virosome had similar characteristics to the parent virus and contained both the hemagglutinin (HA, 60-75 kDa) and neuraminidase (NA, 220 kDa) protein, with preserved biological activity, such as hemagglutination activity. The virosome structure was analyzed by negative stained transmission electron microscope (TEM) demonstrated that the spherical shapes of vesicles with surface glycoprotein spikes were harbored. In conclusion, the biophysical properties of the virosome were similar to the parent virus, and the use of octaethyleneglycol mono (n-dodecyl) ether to solubilize viral membrane, followed by removal of detergent using polymer beads adsorption (Bio- Beads SM2) was the preferable method for obtaining avian influenza virosome. The outcome of this study might be useful for further development veterinary virus vaccines. © 2013 PVJ.