Hydrogels are soft materials constructed of physically or chemically crosslinked polymeric net-works with abundant water. The crosslinkers, as the mechanophores that bear and respond to mechanical forces, play a critical role in determining the mechanical properties of hydrogels. Here, we use a polyprotein as the crosslinker and mechanophore to form covalent polymer hydrogels in which the toughness and fatigue fracture are controlled by the mechanical unfolding of polyproteins. The protein Parvimonas sp. (ParV) is super stable and remains folded even at forces > 2 nN; however, it can unfold under loading forces of ~100 pN at basic pH values or low calcium concentrations due to destabilization of the protein structures. Through tuning the protein unfolding by pH and calcium concentrations, the hydrogel exhibits differences in modulus, strength, and anti-fatigue fracture. We found that due to the partially unfolding of ParV, the Young’s modulus decreased at pH 9.0 or in the presence of EDTA (Ethylene Diamine Tetraacetic Acid), moreover, because partially unfolded ParV can be further completely unfolded due to the mechanically rupture of ester bond, leading to the observed hysteresis of the stretching and relaxation traces of the hydrogels, which is in line with single-molecule force spectroscopy experiments. These results display a new avenue for designing pH- or calcium-responsive hydrogels based on proteins and demonstrate the relationship between the mechanical properties of single molecules and macroscopic hydrogel networks.