The main aim of this study was, using biomechanistic approach, to synthesize and characterize amperometric stable gold nanoparticles (AuNPs) under different pH conditions using UV Spec, dynamic light scattering and TEM with energy dispersive X-ray analysis. The biomolecules involved in conjugation and reduction were further characterized by Fourier transform infrared analysis. The pH stabilized nanoparticles were studied to determine the functional and molecular mechanism of cell death on liver cancer (HepG2) cell line and gastric cancer (YCC3) cell line. The zeta potential and TEM imaging demonstrated that AuNPs were spherical in nature and can pass through the cellular membrane because of their intrinsic properties of AuNPs to bind to carbon-bonded sulfhydryl (-C-SH or R-SH) group and, therefore, could interact with intracellular components of the cell which was confirmed through phase contrast microscopy. Altered molecular mechanism and cellular effects in different cancer cell suggest a potential for in vivo applications of gold nanomaterials.