Centromeres and telomeres are DNA/protein complexes and essential functional components of eukaryotic chromosomes. Previous studies have shown that rice centromeres and telomeres are occupied by CentO (rice centromere satellite DNA) satellite and G-rich telomere repeats, respectively. However, the protein components are not fully understood. DNA-binding proteins associated with centromeric or telomeric DNAs will most likely be important for the understanding of centromere and telomere structure and functions. To capture DNA-specific binding proteins, affinity pull-down technique was applied in this study to isolate rice centromeric and telomeric DNA-binding proteins. Fifty-five proteins were identified for their binding affinity to rice CentO repeat, and 80 proteins were identified for their binding to telomere repeat. One CentO-binding protein, Os02g0288200, was demonstrated to bind to CentO specifically by in vitro assay. A conserved domain, DUF573 with unknown functions was identified in this protein, and proven to be responsible for the specific binding to CentO in vitro. Four proteins identified as telomere DNA-binding proteins in this study were reported by different groups previously. These results demonstrate that DNA affinity pull-down technique is effective in the isolation of sequence-specific binding proteins and will be applicable in future studies of centromere and telomere proteins.