Cancer has been one of the leading causes of death worldwide. Chemotherapy is a proposed therapeutic in recent years; however, its efficiency is restricted due to multiple drug resistance (MDR), toxicity on normal cells, and poor physicochemical properties. The use of natural compounds with less cytotoxicity on normal cells is a promising approach to cancer treatment. Curcumin (CUR) is a plant flavonol from the flavonoid group of polyphenols with anti-tumor activity. Nonetheless, low solubility, poor permeability, and short biological half-life hamper the use of curcumin as an anti-cancer drug. The principal aim of this study was to augment CUR loading as a hydrophobic drug and prolong the release time. Curcumin was loaded into a hydrogel nanocomposite of chitosan (CS)-agarose (AG)-montmorillonite (MMT) to attain this goal. The use of MMT nanoparticles in the CS-AG hydrogel improved the loading efficiency from 49% to 62%. The average diameter of the nanocomposite particles in the FESEM images was within the range of 30 nm. Zeta potential of the hydrogel nanocomposites was 47mV, which demonstrates the good stability of the hydrogel nanocomposites. The inclusion of all components in the nanocomposite was proved through the presence of all of the characteristic peaks of the components in the FTIR spectrum. The drug release profile showed the pH-responsive behavior of CS-AG-MMT hydrogel nanocomposites with extended-release over 96 h. The cytotoxicity of fabricated nanocomposites on the MCF-7 cell line was evaluated. Curcumin-loaded CS-AG-MMT showed significant cytotoxicity compared to the control group (p<0.001) and curcumin as a free drug (p<0.05). The developed nanostructure is a promising vehicle with the potential to enhance curcumin loading and achieve sustained release of curcumin with significant cytotoxicity on MCF-7 cells.