Two-dimensional 2D carbon materials especially graphene has gained a significant potential in biomedical and pathological research over a decade. Graphene or Graphene Oxide (GO) is a new material to the field of clinical nanomedicine which is necessary to correlate the physiochemical properties with the existing nanomaterials. The present study is intended to determine the physiochemical properties of graphene leading to potential molecular or cellular injury. Initially we correlated the cellular responses to the lateral dimensions of GO. For this purpose, nano graphene oxide was synthesized by hummer’s method and obtained fine Nano-GO flakes of 200nm size. The crystalline dimensions, micro strain and dislocation densities of the Nano-GOs were analyzed by using the Powder X-Ray Diffraction (PXRD) and Scanning Electron Microscope (SEM) was used to determine the shape of the as-synthesized Nano-GOs. The cytotoxicity and oxidative stress were evaluated by using the cultured human lung epithelium cells (BEAS-2B). GO nanoflakes were exposed to the cultured cells in a dosage pattern of 1, 10, and 100µg/ml. The interactions of GO nanoflakes with the cultured cells were studied using the analytical techniques confocal microscopy, flow cytometry and Transmission Electron Microscope (TEM). It is found that GO nanoflakes interacted with the plasma membrane and internalized via clathrin and caveolae-mediated endocytosis. From the obtained results we summarize that mechanism of interaction of GO nanoflakes with non-phagocytic cell lines over time that can be exploited for the design of biomedically-applicable 2D transport systems. Keywords: 2D Materials, Graphene, Hummers Method, BEAS-2B, Cytotoxicity and Oxidation Stress.