Bacillus subtilis strain NCD-2 is an excellent biocontrol agent against plant soil-borne diseases. With the purpose of understanding the colonization characteristics of strain NCD-2, firstly, a constitutive expression promoter was cloned from strain NCD-2 and was used to construct GFP-labeled strain NCD-2. The GFP-labeled strain NCD-2 showed strong green fluorescence under planktonic cells and biofilm formation. The colonization characteristics of strain NCD-2 on different parts of cotton root were qualitatively observed by confocal laser scanning microscopy (CLSM). Results showed that strain NCD-2 mainly colonized on the zone of differentiation and elongation. Rhizosphere populations of B. subtilis strain NCD-2 on different cotton root were quantitatively evaluated by traditional plating count and quantitative PCR (qPCR) analysis in both autoclaved soil and non-autoclaved soil, respectively. Results showed that both traditional plating count and qPCR analysis showed similar trend for colonization characteristics of strain NCD-2. The greatest strain NCD-2 populations were in the root tip, at 9.19 x 10(7) CFU g(-1) root and 6.75 x 10(7) CFU g(-1) root as estimated by qPCR in non-autoclaved and autoclaved soil, respectively. This study provides a clearer understanding of the interactions between biocontrol agent and plant, as well as with the indigenous microorganisms in the soil.