In the last decade, gold nanoparticles have emerged as promising agents for in vitro bio-sensing and in vivo cancer theranostics. However, different investigations have reported widely varying cytotoxicity and uptake efficiency of gold nanoparticles depending upon their size. Therefore, more extensive studies are needed to standardize these biological effects as a function of size on a particular cell line. In addition, to obtain robust confirmation on the correlation of a size to biological effect, thorough mechanistic study must also be performed. In this study, the size dependent biological activities of gold nanoparticles on osteosarcoma cells is investigated towards exploring their potential theranostic application in bone cancer, for which very scarce literature reports are available. Tris-assisted citrate based method was optimized to synthesize stable gold naoparticles of 40-60 nm sizes. Nanoparticles were characterized through UV-Vis spectroscopy, field emission scanning electron microscope (FESEM) and dynamic light scattering (DLS). Increasing concentrations of gold nanoparticles (AuNPs) of 46 nm size, enhanced the rate of reactive oxygen species (ROS)-induced apoptosis in MG63 cells by disrupting their mitochondrial membrane potential. Considerably higher cell death was observed for 46 and 60 nm AuNPs compared to 38 nm at all concentrations of 200, 400 and 800 ng/mL. Further, molecular signatures of cellular apoptosis under nanoparticle treatment were optically assessed through surface enhanced Raman scattering (SERS). A significant Raman enhancement in cancer cells under treatment of larger gold nanoparticles (46 and 60 nm) at fixed wavelength of 785 nm and laser power of 8.0 mW was evident. In corroboration with molecular biology techniques, SERS observation confirmed the size-dependent apoptotic phenomena in osteosarcoma cells under treatment of gold nanoparticles. Study demonstrates a facile, non-active targeting approach for detection of size-dependent AuNP-induced apoptosis in osteosarcoma cells through label-free SERS method.