The toxicity of nanomaterial is largely determined by particle size, shape, surface charge, composition, concentration, and surface modifications. Due to their nanoscale dimensions, they can interact with biological systems and induce dose-dependent cellular responses. In this study Carbon nanosphere (CNS), were synthesized by probe sonication method and characterized using X-ray Diffraction (XRD), Fourier-Transform Infrared (FTIR), Raman spectroscopy, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Zeta Potential measurement. The dose-dependent toxicity of CNS was evaluated using Drosophila melanogaster (Oregon K strain), in both larval and adult flies (female and male) were fed with CNS containing diet at varying concentrations (50-200 µg/ml). Behavioural toxicity was assessed through larval crawling and adult climbing assays. Biochemical evaluations included measurement of Reactive oxygen species (ROS) level, Superoxide dismutase (SOD) enzyme activity, protein content, and Glutathione S- Transferase (GST) were performed. Other assays, including survival, haemolysis, MTT, fluorescence studies, along with histopathological studies, were also performed. Overall, CNS exhibits high biocompatibility at lower concentrations. But induces oxidative stress and cytotoxic effect at 200 µg/ml. These findings establish a safe exposure window and highlights the importance of dose-dependent evaluation for biomedical applications.