Aim: Modification of nanomaterials with different synthesis methods can affect their biological response, as well as their use as nanotherapeutics. It is necessary to address and understand the safety issue of these particles through toxicological evaluation with an underlying mechanism of interaction. With the fast entry of aluminum-based nanoparticles into the industry, their potential exposure has also increased significantly. Aluminum oxide nanoparticles (Al2O3 NPs) are among the priority materials by international organizations. Studies have not yet elucidated the toxic response of Al2O3 NPs depending on their size range. Materials and Methods: Therefore, this study aimed to investigate toxicological effects of Al2O3 NPs depending on size range on MCF-10 and MCF-7 cells by WST-1 test, hemolytic activity on red blood cells and irritation effects by HET-CAM test. Results: As a result of tests, all size ranges of Al2O3 NPs didn’t show any cytotoxic effects on MCF-10 and MCF-7 cells, also none of sizes of Al2O3 NPs were caused hemolysis (<2%). It was observed that there was no irritating effect in all size ranges on HET-CAM test. Conclusion: In conclusion, risk assessments in terms of characteristic features as the size of Al2O3 NPs showed that they have the potential to provide safe use in drug delivery systems and immobilization studies.