Abstract: Self-assembly is the basis of the formation of biological macromolecular structure. Enzyme-instructed self-assembly (EISA) with the help of tool enzymes, realizing the conversion of small molecular compounds to supramolecular nanostructures at specific sites, become a new strategy for drug discovery.In recent years, the exploration of EISA for developing malignant cancer therapy and imaging has made considerable progress, achieving the precise regulation and tumor targeting of nanostructures. This paper reviews the latest progress of EISA in the field of tumor diagnosis and treatment, the functions and characteristics of tool enzymes such as alkaline phosphatase, sirtuin, tyrosinase, γ-glutamyltranspeptidase and caspase-3,summarizes the research status of EISA targeting multiple organelles in tumor therapy, and introduces the application of EISA in tumor imaging, aiming to provide reference forthe research of EISA strategy in tumor diagnosis and treatment.