Objectives: It has been estimated that one-third of the world population has been infected by toxoplasmosis. Our goal for this study was to evaluate the diagnostic performance of Nanogold beads-based ELISA in serodiagnosis of human toxoplasmosis and improving identification methods based on detection of circulating Toxoplasma surface antigen grade III (SAG3).  Patients & Methods: This study was conducted on 100 individuals. They were divided into 3 groups: group (I):50 T. gondii infected patients &group (II):30 Patients harboring other parasites than T. gondii which was divided as following, ten patients infected with Schistosoma mansoni, ten patients infected with Giardia lamblia and ten patients infected with Entamoeba histolytica while group (III): 20 Apparently healthy individuals free from toxoplasmosis and other parasitic infections.  Results: Detection of Toxoplasma surface antigen grade III in the serum samples by Nano-sandwich ELISA was higher than sandwich ELISA regarding specificity (96% vs. 84%); PPV (95.7% vs. 85.2%) and diagnostic accuracy (93% vs. 88%). Sandwich ELISA was higher than Nano-sandwich ELISA regarding sensitivity (92% vs.90%) and NPV (91.3 %vs. 90.6 %). In our work, we apply this new technique for the first trial for diagnosis of human toxoplasmosis.  Conclusion: We concluded that the use of gold nanoparticles offers the potential advantage of improving the diagnostic testing of toxoplasmosis based on detection of circulating Toxoplasma surface antigen grade III(SAG3). The novel Nano diagnostic assay significantly increased specificity, PPV and diagnostic accuracy.