This study compared the effects of air freezing (AF), immersion freezing (IF), and ultrasound-assisted immersion freezing (UIF) at different power levels on the physicochemical properties, oxidative characteristics, and the structural and functional properties of myofibrillar protein (MP) of fresh beef. The results showed that UIF treatments generally outperformed AF and IF, with UIF-400 W exhibiting the best performance in maintaining beef quality. Specifically, UIF-400 W mitigated muscle fiber damage by inhibiting ice crystal growth, significantly improved water-holding capacity and color stability, reduced shear force, and effectively delayed protein and lipid oxidation. In addition, UIF-400 W brought the rheological properties, emulsifying properties, water distribution, and gel microstructure of MP closer to those of fresh beef. These findings highlight that UIF-400 W can effectively preserve beef quality as well as the structural and functional characteristics of MP, reduce oxidative damage, and thus become a promising technology for quality improvement of beef during freezing.