Abstract: Influenza vaccines have been produced using embryonated chicken eggs for nearly 70 years. However, this platform has several limitations, making it urgent to develop cell culture-based alternatives for influenza vaccine production. This study established a production and purification process for the H3N2 influenza virus vaccine using MDCK cells in suspension culture. First, H3N2 virus was produced in a small-scale suspension culture, followed by a series of steps including 0.65 μm filtration, 300 kDa ultrafiltration concentration, nuclease treatment, Capto Core 700 and Capto Q chromatography, and virus inactivation, to prepare a monovalent H3N2 influenza virus vaccine. Each step was evaluated for purification efficiency based on the Chinese Pharmacopoeia (2020 Edition) standards, including measurements of host cell DNA content, hemagglutination (HA) titer, total protein content, hemagglutinin content, and Vero cell host protein content. Finally, 4-to 6-week-old BALB/c mice were subcutaneously immunized twice, and sera were collected from tail veins to evaluate immune efficacy using the hemagglutination inhibition (HI) assay. The results showed that the total protein removal rate was (97.94±0.09)%, host protein removal rate was (99.46±0.03)%, host DNA removal rate was (99.99±0.01)%, and hemagglutinin recovery rate was 19.63±2.25%. The final formulated monovalent vaccine met the pharmacopoeia standards. Animal experiments demonstrated that the H3N2 vaccine prepared using MDCK suspension culture exhibited immune efficacy comparable to the egg-based influenza vaccine and had good safety.This study established a production and purification process for an H3N2 influenza virus vaccine using MDCK cells in suspension culture. The resulting vaccine demonstrated good immunogenicity and safety.