Abstract: In this study, the fruiting body of Rhizopogon luteolus that is ecto-mycorrhizal fungi of Pinus tabulaeformis was considered as a separation material and the pure cultures of Rhizopogon luteolus were obtained by tissue culture. Strain inoculated in different culture medium with a reference standard of mycelium growth rate and growth situation. The basic culture medium PACH was selected as a suitable growth of mycelium. Based on this, the cultural characteristics like pH, carbon source, nitrogen source were studied by single factor tests. The research results showed that the pH6.0, glucose, Ammonium tartrate were the best pH value, carbon and nitrogen source respectively. And the growth of mycelium was the fastest in （pHS.0＋beef extract＋ sucrose） combination, the growth situation of mycelium was the best in （pH6.0＋beef extract＋ glucose） combination. By the three optimum level of pH, carbon and nitrogen sources orthogonal tests.