Abstract: 【Objective】Virus-like particles (VLPs ) play a crucial role in research of virology and development of novel vaccines. In this study the authors focus on the major structural proteins which involves in the assembly and release of influenza A VLPs.【Method】A serial of eukaryotic expression plasmids, including the plasmid simultaneously expressing HA, NA and M1 proteins, the plasmid simultaneously expressing HA and NA proteins and the plasmid expressing HA protein of influenza virus, were developed. To determine the key proteins in assembly and release of VLPs, 293T cells were transfected with the plasmids mentioned above, and VLPs were detected by hemagglutinin test and transmission electron microscope observation. 【Result】The result showed that the 293T cells simultaneously expressing HA, NA and M1 proteins, or HA and NA proteins could produce VLPs, while the 293T cells expressing only HA could not. 【Conclusion】In this study, the eukaryotic expression plasmid which could express 3 proteins simultaneously was used to determine the key proteins in assembly and release of VLPs. The results indicated that both HA and NA were crucial in this process, while M1 was not necessary.