MF59 is a squalene-based oil-in-water nano-adjuvant. It has been extensively applied in clinical settings, demonstrating excellent safety and adjuvant effects. In the future, it will play a greater role in the development of vaccines for the prevention of emerging infectious diseases. This paper systematically reviews the immunological mechanisms, non-clinical safety studies, and clinical application progress of MF59. Furthermore, it highlights considerations for pharmaceutical research and evaluation of MF59, offering insights into its potential applications in future vaccine development.