Objectives: Seasonal influenza leads to substantial global morbidity and mortality, especially in adults aged 65 years and older, who present poor immune responses to standard-dose influenza vaccines. This study aimed to systematically evaluate the preclinical safety and immunogenicity of a high-dose quadrivalent seasonal influenza split vaccine (HD-QIV), providing preclinical evidence for its clinical application in the elderly. Methods: Following GLP guidelines, we performed single-dose and repeated-dose toxicity tests in Sprague–Dawley rats, active systemic anaphylaxis assays in guinea pigs, and immunogenicity assessments in young and aged BALB/c mice. Safety indicators included general clinical signs, hematology, blood biochemistry, histopathology and allergic reactions; immunogenicity was evaluated via hemagglutination inhibition (HI) antibody titers and antigen-specific cellular immune responses. Results: HD-QIV only caused mild and reversible local irritation in rats without obvious systemic toxicity, and no dose-related systemic anaphylaxis was observed in guinea pigs. HD-QIV induced robust and dose-dependent humoral immune responses, and showed significantly higher HI antibody titers, earlier seroconversion and longer antibody persistence than standard quadrivalent influenza vaccine in aged mice. Cellular immunity was slightly enhanced but not the dominant protective response. Conclusions: The HD-QIV demonstrates favorable preclinical safety and superior immunogenicity, supporting its further clinical development and use as a priority vaccine for the elderly population.