The characterizations of green synthesized gold nanoparticles (AuNPs) prepared using Ocimum sanctum leaf extract were done by UV-Spectrophotometer at 500-540 nm. The XRD data obtained were found similar to gold JCPDS File No- 04-0784. SEM and TEM analysis of AuNPs revealed spherical shape and size of 200 nm. Further FT-IR data indicated the various biomolecules present in Ocimum sanctum leaf extract provides stability to gold nanoparticles synthesis. The AuNPs were studied for their anti-cancer activity on Dalton’s lymphoma (DL) cells and the results obtained with IC50 value of < 50 ng/ml performed by MTT assay. Further, to confirm anti-tumor potential and the mode of action of the synthesized AuNPs, cell viability assay, nuclear morphology, DNA fragmentation assay, mitochondrial membrane potential (ΔΨm) analysis, and cell cycle analysis were done using DL cells. DL cells treated with the AuNPs showed reduced cell viability, altered nuclear morphology, typical apoptotic DNA ladder formation and apoptosis. From the above finding it can be concluded that the AuNPs have potential to decrease the proliferation of tumor cells and enhanced the production of ROS. Gold nanoparticles used in cancer detection and diagnosis/treatment are mainly in preclinical stages of cancer development.