Isolates from the stroma of Ophiocordyceps sinensis, produced generously ascocarps with reticuloperidium. Morphological characteristics and molecular identification indicated that the repre-sentative strain Pseu-F was Pseudogymnoascus roseus Raillo. The optimum temperatures for mycelial growing were 17.5 °C?20.0 °C. Submerged culture media were screened by means of orthogonal design experiments. Test factors included sucrose, glucose, peptone, yeast extract, potato, soybean, mineral salt and Vitamins. Based on the results of tests, the optimized combinations of culture medium are sucrose 20 g, glucose 10 g, peptone 10 g, yeast extract 5 g, soybean 50 g, potato 100 g, in 1 L liquid medium.