Introduction The accurate diagnosis of toxoplasmosis has critical importance in pregnant women. Nanotechnology and molecular biology are making possible opportunities for accurate and rapid diagnosis of many infectious diseases. Aim and Methods The aim of our study was to compare nano-gold ELISA with ELISA and PCR for diagnosis of toxoplasmosis using Toxoplasma surface antigen grade 1 (SAG1) in pregnant women seeking antenatal care in outpatient clinics. Results PCR showed the highest diagnostic values than nano-gold ELISA and ELISA regarding sensitivity (97.3% versus 89.2% and 83.8%); specificity (100% versus 94% and 88%); and diagnostic accuracy (98.9% versus 91.95% and 86.2%), respectively. There is no statistical difference between PCR and nanogold ELISA results. Discussion Nano-gold ELISA had a significant improvement in diagnosis than the traditional ELISA method. Most likely with the assistance of nanoparticles, more antibodies enter the antigen–antibody complex because of the considerable improvement in the surface area of nano-gold particles. Conclusion Although PCR had higher diagnostic values than nano ELISA, nano ELISA is cheaper and easier than PCR. We recommend nano-gold ELISA with SAG1 as a promising technique in the diagnosis of toxoplasmosis and survey studies.