Targeted drug delivery systems having controlled drug release property with an inherent fluorescence reporter have drawn a lot of attention in nanomedicine. However, only very few prodrugs can be directly used to construct such delivery systems. Herein, we report that an amphiphilic chlorambucil-based prodrug consisting of a fluorescence reporter and a d-mannose targeting ligand could directly self-assemble into glutathione-responsive nanovesicles for selective cancer therapy and intracellular imaging. These nanovesicles could be dissociated to release the chlorambucil drug with obviously red-shifted fluorescence when internalized by d-mannose receptor-overexpressed MCF-7 cancer cells. In addition, the nanovesicles displayed better selectivity and higher therapy efficiency than free chlorambucil drug.