Strain-stiffening properties derived from biological tissue have been widely observed in biological hydrogels and are essential in mimicking natural tissues. Although strain-stiffening has been studied in various protein-based hydrogels, effective approaches for tuning the strain-stiffening properties of protein hydrogels have rarely been explored. Here, we demonstrated a new method to tune the strain-stiffening amplitudes of protein hydrogels. By adjusting the surface charge of proteins inside the hydrogel using negatively/positively charged molecules, the strain-stiffening amplitudes could be quantitively regulated. The strain-stiffening of the protein hydrogels could even be enhanced 5-fold under high deformations, while the bulk property, recovery ability and biocompatibility remained almost unchanged. The tuning of strain-stiffening amplitudes using different molecules or in different protein hydrogels was further proved to be feasible. We anticipate that surface charge adjustment of proteins in hydrogels represents a general principle to tune the strain-stiffening property and can find wide applications in regulating the mechanical behaviors of protein-based hydrogels.