Abstract: In this study, field trials were conducted to examine the effects of cotton stalk biochar on microbial population, function and structural diversity of microorganisms in rhizosphere soil of continuous cotton cropping field in Xinjiang by plate count, Biolog and DGGE methods. The experiment was a factorial design with four treatments: 1) normal fertilization with cotton stalk removed (NPK); 2) normal fertilization with cotton stalk powdered and returned to field (NPKS); 3) normal fertilization plus cotton stalk biochar at 22.50 t·hm^-2(NPKB-1); and 4) normal fertilization plus cotton stalk biochar at 45.00 t·hm^-2(NPKB-2). The results showed that cotton stalk biochar application obviously increased the numbers of bacteria and actinomycetes in the rhizospheric soil.  Compared with NPK treatment, the number of fungi was significantly increased in the NPKB-1 treatment, but not in the NPKB-2 treatment. However, the number of fungi was generally lower in the biochar amended (NPKB-1, NPKB-2) than in the cotton stalk applied plots (NPKS). Application of cotton stalk biochar increased values of AWCD, and significantly improved microbial richness index, suggesting that the microbial ability of utilizing carbohydrates, amino acids and carboxylic acids, especially phenolic acids was enhanced. The number of DGGE bands of NPKB-2 treatment was the greatest, with some species of Gemmatimonadetes, Acidobacteria, Proteobacteria and Actinobacteria being enriched. UPGMC cluster analysis pointed out that bacterial communities in the rhizospheric soil of NPKB-2 treatment were different from those in the NPK, NPKS and NPKB-1 treatments, which belonged to the same cluster. These results indicated that application of cotton stalk biochar could significantly increase microbial diversity and change soil bacterial community structure in the cotton rhizosphere soil, thus improving the health of soil ecosystem.