A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant-microbe interactions in the future. © 2017 Vu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.