Fungal mediated biological synthesis of magnesium oxide nanoparticles (MgO-NPs) is a new approach and eco-friendly technique in context to green nanotechnology. The fungal isolate Aspergillus sp. was used for biosynthesis of magnesium oxide nanoparticles by using Mg (NO3)2.6H2O as a precursor. This fungal isolate was identified on the basis of manual methods of morphological and microscopic investigation. The optimum conditions for pure fungal isolate to biosynthesis of MgO-NPs was grown in a Czapex Dox broth medium at 32 ± 2°C for 6 days, p H 6, and shaking condition at 150 rpm in 100 ml distilled water. Fungal filtrate treated with 3mM of Mg(NO3)2.6H2O at pH8 and kept for 36 h at 35°C.The NPs synthesized showed an absorption peak at 400 nm in UV-Vis spectrum corresponding to the Plasmon resonance of magnesium oxide nanoparticles.