This study presents a mechano-controlled biocatalytic system for hydrogel regulation, utilizing genetically engineered thrombin and its inhibitor hirudin covalently incorporated into hydrogel networks. Mechanical stretching of the hydrogel disrupts the noncovalent inhibitory interaction between thrombin and hirudin, activating thrombin's catalytic activity. This system enables the regulation of hydrogel mechanical properties through reversible on/off switching of thrombin activity. Under cyclic tensile loading, hydrogels exhibit self-stiffening or self-softening behaviors depending on the presence of substrates that self-assemble into new networks or cleavable peptide crosslinkers. The system also demonstrates the programming of bilayer hydrogels to exhibit tailored shape-morphing behavior under mechanical stimulation. The developed system provides 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. The study highlights the importance of enzyme activity regulation through mechanical force, which is essential for controlling cellular metabolic processes and tissue regeneration. The research also addresses the challenges of converting mechanical force into biochemical signals and demonstrates the application of ultrasound and mechanical stretching to control biocatalytic reactions. The findings suggest that the reversible control of enzyme activity at the molecular level can be used to regulate the mechanical properties of hydrogels at the material level, enabling the development of self-stiffening or self-softening hydrogels. The study further demonstrates the potential of bilayer hydrogels for shape-morphing applications, with the ability to transform from 2D structures to 3D objects upon mechanical stimulation. The results indicate that the developed system has broad applications in areas such as drug delivery, tissue engineering, and soft robotics.This study presents a mechano-controlled biocatalytic system for hydrogel regulation, utilizing genetically engineered thrombin and its inhibitor hirudin covalently incorporated into hydrogel networks. Mechanical stretching of the hydrogel disrupts the noncovalent inhibitory interaction between thrombin and hirudin, activating thrombin's catalytic activity. This system enables the regulation of hydrogel mechanical properties through reversible on/off switching of thrombin activity. Under cyclic tensile loading, hydrogels exhibit self-stiffening or self-softening behaviors depending on the presence of substrates that self-assemble into new networks or cleavable peptide crosslinkers. The system also demonstrates the programming of bilayer hydrogels to exhibit tailored shape-morphing behavior under mechanical stimulation. The developed system provides 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. The study highlights the importance of enzyme activity regulation through mechanical force, which is essential for controlling cellular metabolic processes and tissue regeneration. The research also addresses the challenges of converting mechanical force into biochemical signals and demonstrates the application of ultrasound and mechanical stretching to control biocatalytic reactions. The findings suggest that the reversible control of enzyme activity at the molecular level can be used to regulate the mechanical properties of hydrogels at the material level, enabling the development of self-stiffening or self-softening hydrogels. The study further demonstrates the potential of bilayer hydrogels for shape-morphing applications, with the ability to transform from 2D structures to 3D objects upon mechanical stimulation. The results indicate that the developed system has broad applications in areas such as drug delivery, tissue engineering, and soft robotics.