This study presents a mechano-controlled biocatalytic system for regulating the mechanical properties of hydrogels. The system involves genetically engineered thrombin and its inhibitor, hirudin, which are covalently coupled to hydrogel networks. The catalytic activity of thrombin is reversibly switched on by stretching the hydrogels, disrupting the noncovalent inhibitory interaction between thrombin and hirudin. Under cyclic tensile loading, the hydrogels exhibit self-stiffening or self-softening behavior when substrates that can self-assemble into new networks are present, or when thrombin-cleavable peptide crosslinkers are part of the original network. Additionally, bilayer hydrogels are programmed to exhibit tailored shape-morphing behavior under mechanical stimulation. This work demonstrates a proof of concept for mechanically controlled reversible biocatalytic processes, showcasing their potential for regulating hydrogels and proposing a biomacromolecular strategy for mechano-regulated soft functional materials.This study presents a mechano-controlled biocatalytic system for regulating the mechanical properties of hydrogels. The system involves genetically engineered thrombin and its inhibitor, hirudin, which are covalently coupled to hydrogel networks. The catalytic activity of thrombin is reversibly switched on by stretching the hydrogels, disrupting the noncovalent inhibitory interaction between thrombin and hirudin. Under cyclic tensile loading, the hydrogels exhibit self-stiffening or self-softening behavior when substrates that can self-assemble into new networks are present, or when thrombin-cleavable peptide crosslinkers are part of the original network. Additionally, bilayer hydrogels are programmed to exhibit tailored shape-morphing behavior under mechanical stimulation. This work demonstrates a proof of concept for mechanically controlled reversible biocatalytic processes, showcasing their potential for regulating hydrogels and proposing a biomacromolecular strategy for mechano-regulated soft functional materials.