Nowadays, how to improve the selectivity of chemotherapy drugs and reduce their side effects is still a significant challenge for cancer research. Although enzyme-instructed self-assembly (EISA) has provided a promising approach for selective cancer therapy, the application of EISA is still suffering from requiring much higher concentrations for inhibiting cancer cells. Therefore, new strategies are needed to maximize the anticancer efficacy and preserve the selectivity of EISA. In this study, we rationally designed and synthesised a novel peptide-based prodrug molecule, NapGDFDFpYSV, combining EISA with the YSV anticancer peptide. The activity of the prodrug molecule was remarkably reduced by masking "Y" with a phosphoryl (-PO3) group and was recovered through dephosphorylation in situ by alkaline phosphatase (ALP) catalysis. The resulting monomer, NapGDFDFYSV, as a hydrogelator further self-assembled into the nanodrug on the cell surface, resulting in enhanced cellular uptake and selective high cytotoxicity to cells overexpressing ALP via action on histone deacetylase. Moreover, the required cell inhibition concentration of NapGDFDFpYSV was much lower than its critical micelle concentration (CMC), exhibiting outstanding advantages compared with separately used EISA without the anticancer peptide. Our study provides a new strategy to improve the cytotoxicity selectivity and bioactivity of chemotherapy drugs as well as the anticancer efficiency of EISA.