Background: Echinococcus granulosus protoscolex antigen (PSAg) is a protein with significant immunological properties having higher sensitivity and specificity in ELISA. It lowers cross-reaction with antibodies of other parasites and thus its application is recommended in serological diagnosis. Labelling of ELISA with nano-gold particles improved the diagnostic abilities of the laboratory technique in hydatidosis detection. Objective: To evaluate the use of nano-gold dot-ELISA for isolation of E. granulosusPSAg and its application in serodiagnosis of hydatidosis in humans and animals in comparison with dot-ELISA. Material and Methods: Hydatid cyst PSAg was isolated and used for immunization of rabbits to raise IgG polyclonal antibodies (pAb) in antisera. These sera were labeled with horseradish peroxidase (HRP) and used for detection of circulating PSAg in sera of human cases and camels and sheep by dot-ELISA and nano-gold dot-ELISA. Results: Conjugation of the anti-protoscolex pAb with gold nano-particles increased the sensitivity of antigen detection by nano-gold dot-ELISA to 94.4% and specificity to 90%, with positive and negative predictive values of 94.4% and 90%, and an accuracy of detection of 92.9%in both human and animal sera. Conclusion: Nano-gold dot-ELISA technique is more sensitive than dot-ELISA for detection of hydatidosis antigen both in human and animal samples.