Abstract: A wild Lentinus squarrosulus strain（QL-1）,which collected from Zhenning county, Anshun city, Guizhou Province was used as test material.The fruiting body morphology and mycelial ITS sequence analysis were used.The effects of temperature, pH, carbon source, nitrogen source, vitamin, inorganic salt and culture substrate on mycelial growth were studied, in order to optimize the culture medium and culture substrate, and provide reference for further development and application.The results showed that the QL-1 strain was Lentinus squarrosulus,the optimum culture temperature was 30 ℃;and the optimum pH was 5-6;the best carbon and nitrogen sources were fructose and yeast extract, the fastest growth rate of mycelia was 1.07 cm·d^-1 and 0.81 cm·d^-1,respectively; adding maltose and ammonium nitrate in the medium, the mycelia grew thicker; adding proper amount of vitamin B₁,vitamin B₆,manganese sulfate and calcium chloride in the medium could effectively promote the growth of mycelia; adding 20% corncob to culture substrate was beneficial for mycelium growth.