On the growing clinical demands, superparamagnetic iron oxide nanoparticles (SPIONs) have a vital role due to their infinite physical and chemical properties at the nanoscale. The researchers have started to focus more on the magnetite nanoparticle applications with unique shape and size in various filed such as biomedicine, food and environment to synthesize Fe 3 O 4 NP. Here we synthesized SPIONs-Fe 3 O 4 NP successfully by co-precipitation technique. The prepared nanoparticles were characterized using suitable analytical tools for structural, morphological, elemental, optical and thermogravimetric analysis. Moreover, the genotoxicity and hemolysis assay test has been carried out to estimate the toxicity of SPIONs-Fe 3 O 4 NP at various concentrations. The results found that 25 µg/mL concentration of SPIONs-Fe 3 O 4 NP shows good hemocompatibility than other concentrations. The genotoxicity assay was examined in Allium cepa (onion root tips) for chromosomal aberration. Hence, the present study discusses the synthesis characterization, assessing the genotoxicity and hemocompatibility potential for the synthesized Fe 3 O 4 NPs.