Nowadays, molecular methods are needed in a lot of researches. According to molecular markers researches, it was felt the need to high-quality fungal DNA extraction in a simple, low risk, low costs and fast method and at the same efficiency. Conventional techniques for fungal DNA extraction contains use of C-TAB, Phenol, liquid nitrogen, SDS, or DNA extraction kits that which has own advantages and disadvantages. In most fungal DNA extraction methods, it is necessary to use liquid nitrogen to powdering the fungal material. That, this substance is toxic too and also provide and maintenance it has been hard and need special equipment, safety and cost. On the other hand the sample of the fungus should be transferred to the laboratory more precisely without wilting. To do this, you need a freezer at temperatures of -40°C. In this research, it is introduced a simple, repeatable and usable way to fungal DNA extraction. In this method, by doing changes in the amount of centrifuge rounds, amount of material consuming, time and temperature variations, we achieved an optimal method, in respect to time and economics, as well as higher volume extraction and higher-quality DNA. According to this method, at first we separated fungus from the surface of culture medium and in order to dry, we placed inside the open envelope at a temperature of 25-30°C. After powdering hyphae, add extraction buffer and extracting DNA of it, the qualitative tests were performed using electrophoresis and spectrophotometry, the results indicates that was high quality and quantity of extracted DNA. In continuation, by using the ISSR primers, the PCR reaction was performed on it too, that confirmed high efficiency reproduction of it.