H5 subtype highly pathogenic avian influenza virus (HPAIV) of clade 2.3.4.4b has prevailed globally, causing economic losses in the poultry industry and posing zoonotic threats. While inactivated vaccines are advantageous tools for prevention and control, their reliance on egg-based production and live viruses necessitates safer alternatives. Based on multiple design strategies, including signal peptide replacement, transmembrane domain truncation, fusion with Trimer-tag or Helicobacter pylori Ferritin, and adaptation of the EABR [ESCRT (endosomal sorting complex required for transport)- and ALIX (ALG-2-interacting protein X)-binding region] technology, three trimeric hemagglutinin (HA) antigens (S836-Trimer, S836-Ferritin, and S836-ESCRT) were constructed, transiently expressed in HEK293 cells, and the immunogenicity of the candidate antigens was subsequently evaluated through animal studies. Among these, S836-Trimer was identified as optimal based on secretory expression, conformational stability, and immunogenicity. Animal experiments showed that a single immunization with 10 μg of S836-Trimer provided complete protection against a lethal challenge with an epidemic clade 2.3.4.4b virulent strain in chickens, comparable to that of an existing commercial trivalent inactivated vaccine. Subsequently, CHO cell lines stably expressing S836-Trimer were established, achieving ～10-fold higher antigen yield than HEK293 cells and demonstrating the feasibility of large-scale production of the candidate vaccine. Animal trials confirmed the single-dose efficacy of this CHO cell-derived vaccine. Overall, our research indicates that developing candidate avian influenza vaccines based on mammalian cell expression systems is a promising strategy.