Abstract: Bacillus subtilis B006 has been attempted in this study to control wilt disease of cucumber. In order to clear its suppressing mechanism in rhizosphere, population of Fusarium oxysporum f. sp. cucumerinum J. H. Owen (Foc) and B006 in cucumber rhizosphere were determined using real-time PCR. Real-time PCR program of B. subtilis B006 in soil was established for first time with the limit as low as 78.0 CFU g-1. Real-time PCR assays showed that combined use of B006 in substrate, drenching during transplantation and hole application with organic fertilizer can improve its colonization in rhizosphere by 10 times in comparison with its single use in substrate. And its presence resulted in the decline of Foc number over 10 times and 2 weeks delay of the disease development and reduction of disease severity in comparison with the non-B006 treatment. Greenhouse test showed that the addition of B006 powder in substrate can control cucumber wilt effectively with the control efficacy of 53.6%, combined use of 30 mL B006 suspension by drenching at the concentration of 106 CFU g-1 with hole application of 10 g organic fertilizer per seedling during transplantation resulted in the best control performance of 72.2%, while single use of organic fertilizer only resulted in the control performance of 29.4% in comparison with blank control. This study improves our understanding for the interaction of B006 and Foc in cucumber rhizosphere further and provides us a suitable application way of B. subtilis as a bioagent for cucumber wilt management.