Invasive aspergillosis (IA) is an important cause of morbidity and mortality among immunocompromised people. Imaging and specimen tests used in the clinical diagnosis of aspergillosis with weak and indistinct defects leads to delay in the treatment of early aspergillosis patients. The developing molecular techniques provide a new method for the aspergillosis diagnosis. However, the existing methods are complex, time-consuming and may even be potentially hazardous. In this study, we developed a simple and rapid Aspergillus fumigatus spores DNA isolation assay using synthesized zinc oxide (ZnO). ZnO nanoparticles were used to take the place of the traditional commercial lysis buffer. The quality and quantity of the extracted DNA were sufficient for further diagnostics with polymerase chain reaction (PCR) analysis. This method offers easy, green, and economic alternative DNA isolation for the diagnosis of invasive aspergillosis.