The low efficiency of protease hydrolysis on wool keratin limits the development of protease-based wool shrink-proofing finishing. In this paper, a highly efficient protease catalytic system consisting of a trihydroxy organ phosphine compound and the protease Savinase 16L was used for wool shrink-proofing treatment by multiple padding. The directional friction effect, area shrinkage, fineness, and strength of wool samples were measured. Scanning electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, zeta potential, and amino acid analyses were used to characterize the structure of the wool samples. Additionally, the dyeing properties of the treated wool were also investigated. The results showed that the hydrolysis of wool fiber scales using the protease catalytic system was an uneven process from point to surface. The padding treatments could promote a synergistic process of biochemical keratin degradation and physical scale removal, improving the efficiency and uniformity of the reaction. The area of the treated wool sample shrank by 1.65% after the TM31 5 × 4N test, while the tenacity decreased by 9.69%. The isoelectric point of the treated fiber increased from 4.37 to 6.09. Under dyeing conditions, the positively charged surface of the treated wool increased, which significantly increased the adsorption rate of the fiber for negatively charged dyes. In addition, the increase in the surface roughness and surface dyeing seat resulted in a significant increase in the color depth of the treated wool fibers after dyeing. This study provides a feasible route for the industrial application of the protease method for the shrink-proofing treatment of wool.