Sarcoplasmic protein (SP), a water-soluble fraction naturally lost during post-ripening and industrial thawing. This study revealed the concentration-effect mechanism of SP in regulating the gel properties of beef mince by controlling the addition amount of SP (0%–0.5%, m/m). Compared with the control group, the incorporation of 0.3% SP maximized gel strength and water-holding capacity (95.33%) while increasing cooking yield. Dynamic rheology confirmed a higher storage modulus, indicating an elastic-dominant network. Raman spectroscopy revealed a shift from α-helix to β-sheet, while scanning electron microscope observation showed a denser, more uniform microstructure with reduced pore size. The results of the chemical force analysis revealed a significant enhancement of hydrophobic interactions and disulfide bond formation, which collectively drove the elevation of β-sheet relative content in the protein structure. Excess SP (> 0.4%) formed loose aggregates that impeded myosin cross-linking and weakened the gel. Therefore, controlled SP re-addition can improve the beef minced gel property. This study provides a theoretical basis for the intensive processing and utilization of SP and the quality improvement of beef gel products.