Aim: Colloidal gold nano-particles have been used in diverse biomedical applications due to their unique surface, electronic and optical properties. Because of the strong and size-tunable surface Plasmon resonance, fluorescence and easy-surface functionalization, Gold Nanoparticles (AuNPs) have been widely used in biosensors, cancer cell imaging, photothermal therapy, and drug delivery approach. In general, the toxicity of gold nanoparticles depends on their physical dimensions (such as size and shape) and surface chemistry (such as coating). However, functionalized gold nanoparticles show obvious cytotoxicity. To clarify these problems, the cytotoxicity of gold nanoparticles in human blood cells (1st target during exposure) has been studied in the present experiment. Materials and Methods: Red Blood Cells were exposed with the Gold Nanoparticles (AuNPs) in a dose and time dependent manner in vitro. The level of oxidative stress biomarkers viz., Glutathione Reductase (GR), Glutathione Peroxidise (GPx), Glutathione S Transferase (GST), total thiols and malondialdehyde were determined. AuNPs were characterized with the help of spectrophotometer, FTIR, DLS technique, XRD, SEM EDx. Results: The current study was focused on investigating the effect of AuNPs on RBC. The results of our study demonstrated an altered activity of various oxidative stress markers in a time dependent and dose dependent manner. Malondialdehyde, a pro-oxidant and a by-product of lipid peroxidation was found to be significantly increased at differential degrees up on exposure to AUNPs for various time periods. While the MDA levels were increased, a concomitant decrease in the antioxidant markers was observed. Total thiols, Glutathione reductase, Glutathoone S-transferase and glutathione peroxidase were significantly decreased at variable degrees. Effect of AUNPs was evident within 6 hr of exposure, 18 and 24 hr exposure resulted in a much greater increase that could be toxic to the cell. Conclusion: Surface negativity and small size of the AuNPs may be assumed to affect the biochemical and cellular architecture of the organs. Further molecular level analysis will reveal the usability potential of AuNPs in body system for different purposes.