Nanoparticles (NPs) with responsive modalities in biological microenvironments and external stimuli have received great attention as highly efficient and precise cancer therapy agents. In this study, tumor intracellular microenvironment-responsive NPs co-assembled from poly(ethylene glycol)-poly(aspartic acid) [PEG-P(Asp)] copolymer, doxorubicin (DOX), and superparamagnetic iron oxide NPs (SPIONs), termed as PEG-P(Asp)/DOX/SPIONs, were prepared for tumor intracellular microenvironment (enzyme and pH)-responsive and magnetically targeted chemotherapy. The NPs exhibited not only enzyme-responsive degradation in the presence of protease, but also triggered release of DOX at pH 5, which is an acidity similar to endolysosomal microenvironments in tumor cells. Furthermore, the PEG-P(Asp)/DOX/SPIONs showed a contrast effect in magnetic resonance imaging. In vitro viability assays showed that PEG-P (Asp)/DOX/SPIONs could effectively augment the cytocompatibility of DOX compared to free DOX without a change in magnetic forces. Fluorescence microscopy images indicated that the fabricated NPs efficiently increased the targeted uptake and release of DOX within cells. Overall, this hybrid NP system could be a favorable biomedical agent for effective tumor-targeted anti-cancer therapy.