Interactions between DNA and proteins are critical to almost all cellular functions, and the identification of proteins that bind to a DNA sequence of interest (gene-centered approach) is a widely studied area. However, a limited number of gene-centered methods have been developed. In the present study, we built a protein capture technology based on biolistic transformation (PCaT) to capture proteins bound by a target DNA. A promoter sequence or a DNA fragment was labeled with biotin and introduced into the host plants using biolistic transformation. The DNA and its bound proteins were cross-linked using formaldehyde in the transformed plants, and the labeled DNAs were captured using streptavidin magnetic beads, which enabled harvesting of the DNA-bound proteins. After mass spectrum analysis, the proteins associated with the target DNA were identified. Using this method, we identified the upstream regulators of a WRKY gene from Populus davidianaƗP. bolleana. The reliability of the identification of the upstream regulators of WRKY was confirmed using chromatin immunoprecipitation and electrophoretic mobility shift assays. Taken together, the results showed PCaT provides a novel DNA and protein interactome discovery strategy. In addition, based on PCaT, a quick procedure to determine the upstream regulators of a gene of interest was proposed.