With the advancement of precision medicine, there is an urgent demand for in situ imaging and targeted therapy with high specificity and high sensitivity. Traditional fluorescent probes are often limited by aggregation-caused quenching effects and frequently suffer from issues such as high background signals, poor photostability, and insufficient specificity. Aggregation-induced emission(AIE) materials, with their unique characteristic of being “off”at the molecular level and “on” in the aggregated state, provide a new strategy for constructing high-performance biological fluorescent probes. Enzymes, serving as key biomarkers in disease processes, exhibit substrate specificity and high efficiency in biocatalytic reactions, making them an ideal trigger mechanism for achieving controllable response, precise fluorescence activation, and signal amplification at disease sites using AIE materials. This review comprehensively reviews the latest research progress in the interdisciplinary frontier of enzyme-responsive AIE materials for novel diagnostic and therapeutic strategies in biomedicine. Based on the physicochemical nature and structural characteristics of aggregates formed after enzyme response, we systematically categorize three molecular design strategies, with a focus on elucidating their design principles, response mechanisms, and significant achievements in the diagnosis and treatment of various major diseases, including tumor imaging and intraoperative navigation, diagnosis and treatment of bacterial and viral infections, diagnosis of neurodegenerative diseases, and theranostics. By enabling controllable “off-on” signal switching and targeted amplification, enzyme-responsive AIE materials demonstrate great potential in improving imaging signal-to-noise ratio, enhancing treatment precision, and achieving synergistic diagnosis and therapy. Finally, we analyze the current challenges in this field and provide an outlook on future development trends, particularly new design paradigms integrated with artificial intelligence.