Legumain is the only acidic asparaginly-endopeptidase in mammals that is highly up-regulated in tumor tissue and tumor associated cells. In this study, a novel legumain protease-activated micelle was successfully synthesized and prepared by loading with doxorubicin (DOX). The prepared micelle exhibited a spherical morphology and possessed a low critical micelle concentration of 1.21 × 10-3 mg mL-1 with a DOX loading capacity and entrapment efficiency of 4.05% and 60.6% respectively. The release profile of DOX from this micelle formulation was observed to be legumain concentration dependent. The micelle encapsulation of DOX highly enhanced the cellular uptake of DOX by tumor cell lines of DAOY, Y79, MCF-7, and MCF-7/DOX. Furthermore, encapsulation of DOX boosts the cytotoxicity against the tumor cells while reducing cytotoxicity against RPE and HEK293 cells. In addition, blank micelles did not exhibit any biological effects on tumor or RPE or HEK293 cells at the concentration range of 0-300 μg mL-1, indicating good biocompatibility. The results suggest that this micelle formulation has potential applications in sustained drug delivery for legumain up-regulated tumors.