This study reports optimized conditions for the green synthesis of iron (II,III) oxide nanoparticles (FeO NPs) from leaf extract. The synthetic parameters like concentration of leaf extract, solvent system, buffer, electrolyte, pH, and time were optimized for FeO NPs synthesis. FeO NPs were obtained from the synthesis protocol by measuring size (80 3 nm approx.), characteristics color changes, and an absorption peak between 270 nm and 280 nm using a UV-visible spectrophotometer, scanning electron microscope (SEM), and an energy dispersive X-ray spectrometer (EDS) study. Peroxidase activity was tested with 3,3,5,5-Tetramethylbenzidine (TMB) oxidation in the presence of hydrogen peroxide and dye removal activity was tested with malachite green (MG). The results indicated that the successful synthesis of FeO nanoparticles using aqueous leaf extract of is a practical alternative for biomedical applications due to its potent peroxidase activity and high dye removal capacity (about 93% with UV light and 55% with room light).