Stimulus-responsive materials have been widely studied and applied in biomedical fields. Under the stimulation of enzymes, enzyme-responsive materials (ERMs) can be triggered to change their structures, properties and functions. Herein, natural enzymes act as the endogenous trigger. Owing to the specificity of natural enzymes, ERMs can exert functions in the specific tissues containing these enzymes, while remaining inert in other tissues. This is beneficial for modulating the therapy efficacy and alleviating systemic or local toxicities when ERMs are used to deliver therapeutic molecules. This article focuses on introducing enzyme-responsive strategies, ERMs and their applications in cancer and cardiovascular disease diagnosis, therapy and theranostics. Enzyme-responsive strategies provide a promising research cue to construct intelligent biomaterials for disease treatment and diagnosis.