In this study, a new yeast-like fungal was obtained from leaf surfaces collected from Kheyroodkenar forest, Mazandaran, Iran. The properties of this strain, such as morphology, DNA molecular, and product showed that it is related to Aureobasidium pullulans family and named A. pullulans MG271838. The pullulan production conditions by this strain were optimized using a Box-Behnken design. The results showed that the optimum production yield (37.55 ± 0.45 g/l) was obtained in pH of 6.76, sucrose concentration of 6%w/v and yeast extract concentration of 0.2%w/v. The pullulan had a concentration-dependent flow behavior, amorphous structure based on XRD pattern and high thermal stability (decomposition temperature of 300 °C). Also, the chemical structure of pullulan was confirmed by FTIR spectroscopy. In addition, there was a direct relationship between pullulan yield and the gene expression of fks, pgm and ugp as the most important genes in pullulan production.