Abstract: In this paper, two strains of laccase producing fungi Z45 and H53 were isolated by selective medium separation and specific substrate screening from the shrub soil of Salsola laricifolia and Reaumuria soongorica in arid region. The ITS sequences were amplified for molecular identification of the two strains. The effects of temperature, carbon source, nitrogen source, carbon to nitrogen ratio（C/N） and pH on the growth of strain Z45 were studied, on the basis of which, orthogonal experiment was designed to optimize the medium. The results showed the thermotolerant strain Z45 belonged to Acrophialophora sp., with a wide range of temperature adaptation and the optimum growth temperature was 40 ℃. The optimal medium combination was as follows: maltose and ammonium nitrate were used as carbon and nitrogen sources, and the ratio of carbon source to nitrogen source was 15:1,with a pH value of 6.0. A thermotolerant laccase producing ascomycetes Acrophialophora sp. with high application value was obtained from the desert soil of the arid area, which enriched the germplasm resources center of laccase producing microorganisms.