Soil microbe has an important role in forest land. In this study, methods of conventional culture, identifying fatty acid and PCR-DGGE were utilized to analyze soil microbial quantity, fatty acid and bacterial DNA segment in Tarim desert highway forest land, which can provide theoretical foundation for managing Tarim desert highway shelter-forest scientifically. The main results are: construction of Tarim desert highway shelter-forest promoted soil microbial development, and with increase of plantation time of the shelter-forest, the quantity, fatty acid diversity index of soil microbe and diversity index of bacterial DNA segment enhanced notably. As to soil microbial community structure, bacteria are dominant population with amounting for over 80% of total soil microbe, and fungi is not more than 1% of total soil microbe, but soil microbial community structure was different among three soil layers. The conclusions from study methods of conventional culture, biological marker and molecule biology were almost identical, which illuminates that construction of Tarim desert highway made soil microbial activity enhance and so promote nutrient cycle and utilization of Tarim desert highway shelter-forest land.