Clinical success of chemotherapeutic drugs is marred by their non-specific distribution in the body leading to side effects. Thus a drug delivery method directing chemotherapeutic drugs exclusively into cancer cells is needed to increase the therapeutic index. In recent times, bio-catalyzing polymeric nanocarriers have demonstrated great promise for targeted delivery of chemotherapeutic drugs. On similar lines, in the present study, we have developed a diselenide-containing bio-endogenous stimulus (redox and pH) responsive polymeric material by covalently conjugating the pharmaceutical grade gelatin protein with 3’-3’ diselenodipropionic acid (DSePA) through amide linkage as confirmed by various spectroscopic analyses. Further, diselenide (Se-Se) containing protein matrix was used to entrap a wide-spectrum anticancer drug doxorubicin (Dox) and to prepare nanoparticles by nanoprecipitation technique. The optimized reaction condition yielded spherical nanoparticles of size 179 ± 10.5 nm with the corresponding ζ- potential value of -11.53 ± 0.59 mV. The bio-catalyzing behavior of polymeric nanocarrier was evaluated by following the release of entrapped Dox under acidic (pH 5.5) and reducing conditions (1 mM DTT / 10 mM GSH). The results clearly indicated that Gprotein-Se-Se nano-matrix catalyzed the significantly higher release of Dox under acidic and reducing environments. Since tumor cells exhibit acidic (low pH) and reducing (high GSH level) environments as compared to normal cells, the drug is expected to be released specifically within the tumor cells. Cellular studies indeed confirmed the higher uptake as well as cytotoxicity of Dox delivered through Gprotein-Se-Se nano-matrix in lung cancer cells demonstrating its potential utility in targeted chemotherapy.