Influenza viruses cause annual seasonal epidemics and occasional pandemics. Vaccination is most effective at preventing influenza virus infections. Suspensions of the Madin-Darby canine kidney (MDCKs) cell line are used to manufacture cell-based influenza vaccines. In this study, we tested and optimized a range of culture conditions for a chimeric recombinant influenza virus rA/B-H3 in MDCK suspension cells, revealing that the optimal inoculation conditions were multiplicity of infection of 0.001, cell concentration of infection of 4.0 × 10⁶ cells/mL, addition of 2 μg/mL of L-1-tosylamide-2-phenylethyl chloromethyl ketone, time of harvest of 72 h. The optimal parameter setting in a 5-L bioreactor was 50 % dissolved oxygen and pH 7.2 ± 0.05. The rA/B-H3 has stability genetic after 15 passages in MDCKs. The rA/B-H3 had several immune responsesin mice. Hemagglutination inhibition (HAI) antibodies, micro-neutralizing (MN) antibodies, and IgG antibodies were induced in immunized mice, and the mucosal IgA antibody responses were detected in their lung lavage fluids. The IFN-γ-secretion and IL-4-secretion by the mouse splenocytes were induced after stimulation with the specific H3N2 HA protein. Immunized mice resisted the lethal challenge with a wild-type H3N2 influenza virus. This study demonstrated the reliability of the MDCK suspension cell platform for the production of the chimeric H3N2 candidate vaccine, and the rA/B-H3 candidate vaccine is a potentially safe efficial vaccine.