Abstract: As an important cash crop, cotton usually continuously cropped for a long-term, which causes an imbalance of soil microbial ecology, incidence of soil-borne disease increase, yield and quality decline, preventing the healthy development of cotton industry. Biochar application can increase soil microbe activity due to its higher porosity, huge specific surface area and ability of nutrient absorption. Biocontrol agents have been found to be effective in adjusting soil environment, alleviating continuous cropping obstacles and controlling soil-borne diseases. However, it is not clear how the biochar affects soil microbe of continuously cropped cotton field under the application of the biocontrol agent of antagonistic actinomycetes. In this paper, a lab incubation experiment was conducted to study the effects of cotton stalk biochar application on soil microflora of continuous cotton cropping under use of antagonistic actinomycetes. Based on antagonistic actinomycetes application, the experiment was designed in 4 treatments of biochar rate-0 g·kg^-1 (control), 25.0 g·kg^-1, 50.0 g·kg^-1 and 100.0 g·kg^-1. The soil microflora was investigated after 15 days of incubation. The results showed that:1) cotton stalk biochar influenced soil microflora under the application of antagonistic actinomycetes. Compared with control, cotton stalk biochar significantly increased the number of bacteria, actinomycetes and fungi in the soil. Applications with 25.0 g·kg^-1 biochar increased bacteria/fungi (B/F) and actinomycetes/fungi (A/F) by 5 271.2% and 30.8% (P < 0.05), respectively. 2) The number of antagonistic actinomycetes significantly increased with increasing application of cotton stalk biochar. Applications with 100.0 g·kg^-1 biochar into the soil significantly increased antagonistic actinomycete population by 2 672.8% (P < 0.05). Cotton stalk carbon served as a potential carrier of biocontrol actinomycetes. 3) Antagonistic actinomycetes with cotton stalk biochar changed the number and proportion of dominant microorganisms in the soil, and especially improved the number and proportion of Bacillus. The number and proportion of Streptomyces under the treatment with 100.0 g·kg^-1 biochar into the soil were significantly higher than those of control treatment, while the number and proportion of Micromonospora were lower. The number of Aspergillus oryzae, Aspergillus niger and Trichoderma koningii increased while the proportions decreased. Therefore, the application of antagonistic actinomycetes and cotton stalk biochar increased the number of biocontrol agent and enhanced disease-controlling and growth-promoting abilities of antagonistic actinomycete, and also improved the structure of soil microbial community. It was concluded that the application of antagonistic actinomycetes and cotton stalk biochar had the potential to alleviate the adverse effects of continuous cropping cotton.