Objective In order to study the colonization rule of Bacillus subtilis biocontrol strain SEM-9 in the rhizosphere of crops and its influence on the microbial diversity of rhizosphere soil. Method The strain SEM-9 was labeled with green fluorescent protein by natural transformation method, and the colonization in rhizosphere soil, on root surface and in root tissue were observed by inverted fluorescence microscope. The changes of microbial diversity in rhizosphere soil treated with the strain SEM-9 were analyzed by high-throughput sequencing using soil with soilborne diseases as test material. Result The recombinant strain SEM-9-pGFP₂₂ stably expressing green fluorescent protein was constructed. The observations of fluorescence microscopy showed that SEM-9-pGFP₂₂ could colonize on the rhizosphere soil and root surface, but not in root tissue or cell. After treated with the SEM-9 suspension, the incidence rate of cucumber soilborne disease significantly reduced, and the fungal diversity in rhizosphere soil increased. Conclusion The GFP labeling method of SEM-9 strain was successfully established, and the rhizosphere colonization rule of the strain and the control effect on cucumber soilborne diseases were clarified, which lays a foundation for the later development of alternative microbial fertilizer.