The invention discloses a quadrivalent influenza virus subunit vaccine and a preparation method thereof, wherein virus protein after lysis is further purified by using a lysis agent and a new purification method to prepare the tetravalent influenza virus subunit vaccine, the content of four influenza hemagglutinins such as influenza A virus H1N1, influenza A virus H3N2 and two kinds of influenza Bviruses in each dose of the vaccine is more than 80%, and the quadrivalent influenza virus subunit vaccine does not contain adjuvant and does not contain thimerosal and other preservatives. The invention further provides a preparation method of the quadrivalent influenza virus subunit vaccine, wherein the preparation method comprises: virus inoculation, virus proliferation culture, allantoic fluid harvesting, clarification, ultra-filtration concentration, inactivation, lysis and ultracentrifugation purification, gel filtration chromatography purification (ultra-filtration), blending, filtration sterilization, sub-packaging, packaging and other steps. According to the present invention, the quadrivalent influenza virus subunit vaccine can improve the safety of the influenza vaccine, can eliminate the adverse reaction caused by the adjuvant, and can eliminate the toxic-side effects caused by thimerosal.