Enzymes are important macromolecular biocatalysts that accelerate chemical and biochemical reactions in living organisms. Most human diseases are related to alterations in enzyme activity. Moreover, enzymes are potential therapeutic tools for treating different diseases, such as cancer, infections, and cardiovascular and cerebrovascular diseases. Precise remote enzyme activity regulation provides new opportunities to combat diseases. This review summarizes recent advances in the field of enzyme activity regulation, including reversible and irreversible regulation. It also discusses the mechanisms and approaches for on-demand control of these activities. Furthermore, a range of stimulus-responsive inhibitors, polymers, and nanoparticles for regulating enzyme activity and their prospective biomedical applications are summarized. Finally, the current challenges and future perspectives on enzyme activity regulation are discussed.