Compared to the conventional anti-cancer drugs, the nano-drug delivery systems (NDDS) shows a comparatively ideal therapeutic efficacy ascribing their high tumor-target capacity and efficient drug release ability. In this study, a novel macromolecular prodrug conjugate was designed and developed that is passively triggered by the acidic environment of the tumor cells and can release doxorubicin (Dox). The susceptibility of the system to the acidic environment is due to the acid volatile Schiff base covalent bond between grafted carboxymethyl chitosan (CMCs) and Dox. The chemical structure of the finalized system was characterized by 1H-NMR spectroscopy. Because of the hydrophobicity of the Dox and the hydrophilicity of the CMCs, the amphipathic polymeric drug conjugates can self-assemble into the nanoparticles in aqueous solution. The morphology and size of the nanoparticles were observed with TEM (transmission electron microscopy) and DLS (dynamic light scattering). The in vitro drug release studies demonstrated that the drug release rate of the prodrug were visibly higher in acidic conditions (pH 5.0) compared to higher pHs like 6.5 and 7.4. DLS results showed that the mean size of the prodrug nanoparticles increased as the pH of the solution decreased from 7.4 to 6.5 and 5.0. Furthermore, the cell counting kit-8 (CCK-8) assays showed that the polymeric drug nanoparticles had a cytotoxic effect on SKOV3 ovarian cancer cells while the segments of the DDS revealed comparatively lower cytotoxic effect. Moreover, confocal laser scanning microscopy (CLSM) showed that the prodrug could efficiently deliver and release Dox in the nuclei of the cancer cells while this was not observed for the Dox pure drug. These results proved that the carboxymethyl chitosan-CBA-Doxorubicin NPs have superior stabilities, low toxicities, proper sizes, and enhanced curable efficiency for in vitro controlled release. Thus, this pH-sensitive prodrug could be a potential treatment for cancer therapy in the near future.