In this study, self-assembled nanostructure of casein (NSCS) of spherical shape was prepared using an optimized desolvation method with a mean size of 45 nm. Curcumin (Cur), a potent natural antioxidant, an anticancer and poorly soluble agent in aqueous solution, was conjugated to protein nanostructure (NSCS-Cur) using CDI chemical activation method. We further, evaluated the physicochemical stability as well as the biological activity of NSCS-Cur in vitro. When the antioxidant activity (free radical scavenging activity) and cellular antioxidant activity (CAA) assay of nano-formulated curcumin (NSCS-Cur) was evaluated, it revealed that antioxidant activity of NSCS-Cur was higher than that of free Cur in both. Furthermore, we showed that the cytotoxicity of nano-formulated curcumin (NSCS-Cur) was found higher in four different cancer cells (breast cancer; MCF-7 and MDAMB231, cervical cancer; HeLa, osteosarcoma; MG 63) than that of free curcumin. Moreover, the conjugation with folic acid with NSCS-Cur (NSCS-Cur-FA) demonstrated enhanced cytotoxicity in cancer cells than un-conjugated one. When we studied the storage stability and bioactivity, NSCS-Cur formulation showed significant stability (up to 30 days) with ignorable loss in its bioactivity. Hence, we concluded from our findings that curcumin loaded protein nanostructure (NSCS-Cur) might be used to achieve higher stability, enhanced dispersibility in suspension as well as improved bioactivity of curcumin which also can be used in the pharmaceutical application.