There are many methods to diagnose Leishmania spp. a microscopic, serological or molecular methods. However, finding a fast and accurate diagnostic assay is a necessary and urgent requirement nowadays. here, we improved nano detection method to diagnose Leishmania spp. In blood samples. In this study, we used a probe of oligonucleotide sequences associated with AuNPs (20 nm) to diagnose different Leishmania spp. in blood samples of dogs. For this purpose, three series replicates of 10 μl of 23 ng / μl of Leishmania spp. DNA are used. A change in the solution colour to red is an indicator of a positive result, while the change to purple is a negative result. Then we compared these results with the molecular assay of (IST1) gene. The overall relative sensitivity of the result is (90%). The result shows that 10 out of 10 samples of visceral Leishmania are positive with relative sensitivity and specificity (100%). Eight out of 10 samples of cutaneous leishmania are positive with relative sensitivity and specificity (80%). The present method is a fast and accurate method for detecting small amounts of DNA, and it is easy to distinguish by visual inspection.