The functional characterization of novel transcription factors identified by systematic analysis remains a major challenge due to insufficient data to interpret their specific roles in signaling networks. Here we present a DNA-binding sequence discovery method to in vitro identify a G-rich, 11-bp DNA-binding motif of a novel potential transcription factor AtYY1, a zinc finger protein in Arabidopsis, by using polymerase chain reaction-assisted in vitro selection and surface plasmon resonance analysis. Further mutational analysis of the conserved G bases of the potential motif confirmed that AtYY1 specifically bound to these conserved G sites. Additionally, genome-wide target gene analysis revealed that AtYY1 was involved in diverse cellular pathways, including glucose metabolism, photosynthesis, phototropism, and stress response.