Highly pathogenic avian influenza viruses (HPAIVs), including H5N6 strains, pose threats to the health of humans and poultry. Waterfowl play a crucial role in HPAIVs. Since current influenza vaccines induce poor antibody titer in waterfowl, to develop an efficient vaccine against H5N6 infection is in urgent need. In this study, we composed two H5N6 virus-like particles (VLPs) composed of matrix-1 (M1) and hemagglutinin of wild-type (HA-TM) or hemagglutinin with transmembrane domain replacement (HA-TMH3) (designated as H5N6 VLPs-TM and H5N6 VLPs-TMH3). Biological characteristics of the composed H5N6 VLPs were compared including localization, expression, contents of HA trimers, thermal stability, morphology and immunogenicity in Muscovy ducks. Our results indicate that the H5N6 VLPs-TMH3 contained more HA trimers and presented better thermal stability. Moreover, Muscovy ducks immunized with H5N6 VLPs-TMH3 produced higher titer of HI antibody and IFN-γ compared with those immunized with the same dose of H5N6 VLP-TM, thus providing a promising approach for the development of influenza viruses vaccines for waterfowl.