In the current study, biosynthesis of gold nanoparticles was carried out using jellyfish nematocyst venom protein extracted from phosphate-buffered saline (PBS) and double distilled water (DDW). The results of the two different gold nanoparticles reported with spherical morphology with a size range of 31 nm and 27 nm respectively. The toxicity of gold nanoparticles was analyzed using normal Human embryonic kidney 293 (HEK293) cell line by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. In addition, the anti-cancer activity of venom protein, PBS/DDW extracted venom protein-mediated synthesized gold nanoparticles, and standard drug 5-Fluorouracil was evaluated using Michigan Cancer Foundation-7 (MCF-7) cancer cell line.