DPPH is one of the most widely used tests with scavenged-free radical effect, in which the color turns to yellow when tested onto a free stable dark-purple tulip, where natural antioxidants are returned by an electron and a proton. The optical, structural properties of Al2O NPs prepared by the pulsed laser ablation (PLA) Nd: YAG laser method at various energies (500,800,1000 mJ) were studied using color absorbance, UV–VIS spectroscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The results showed that the average particle size is less than 41 nm. Spectroscopic analyses were used to study the antioxidant activity of Al2O3 nanoparticles by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical. The DPPH displacement was shown to be directly proportional to the high energies according to the results. The increased energy of (500, 800 and 1000 mJ) for free radicals are at (64.53 ± 5.487)%, (74.00 ± 2.887)% and (84.67 ± 4.372)% respectively, as compared to ascorbic acid. Furthermore, the toxicity of these nanoparticles on human blood parameters was studied using the complete blood count (CBC) (in vitro) by hematological parameters (PLT), (HGB–Hb), (RBCs), (WBCs), and count type white blood cells, and are compared to control groups. Our results demonstrate no significant changes in levels of (PLT), (HGB–Hb), (RBCs), (WBCs), and count type white blood cells between test and control groups. This data suggests that aluminum oxide nanoparticles have no harmful effect on hematological parameters (in vitro).