A pullulans classified under the family Dothideales. In present study different strains isolated from nature and the best strain with a large amount of pullulan production (51) is selected for optimization. Amount of pullulan and its purity determind by pullulanase enzyme activity. Sucrose and glucose select as source of carbon and this strain produced 40 g/l pullulan on sucrose. The best yield was obtained on 5% sucrose. Production of pullulan under different nitrogen source (yeast extract, ammonium sulphate, sodium nitrate), pH (4- 9), temperature (25, 28, 32 °C) and shacking (120,180,200 rpm) by this strain were investigated. Under the best condition 48g/l pullulan was produced within 5 days. A small pigment in the medium was observed during the. fermentation.